Aloe Preparation For Skin Enhancement

ABSTRACT

Methods for providing skin enhancement and pain relief to an individual in need of treatment by administering to such an individual an effective amount of an immunostimulatory Aloe vera derived composition. Oral and topical methods and compositions are provided. Aloe vera derived immunostimulatory compositions and methods of producing such compositions are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Provisional Patent Application No. 60/928,924, filed May 11, 2007, thedisclosure of which is hereby incorporated herein by reference.

BACKGROUND OF THE INVENTION

In one aspect, the present invention relates to the use of bioactivecompositions produced, isolated, extracted, based on or derived fromeither an Aloe vera plant or a portion thereof, for skin enhancement andfor pain relief. The invention also relates to immunostimulatorycompositions, stabilized immunostimulatory compositions, and methods ofmaking and using such compositions.

The genus Aloe (Liliaceae) is a shrubby tropical/subtropical plant whichhas succulent and elongated leaves. Of the more than 360 Aloe speciesknown, Aloe barbadensis Miller (Aloe vera Linne) is the most widelyused, both commercially and for its therapeutic properties. Aloe veraplants contain two major juice materials: first, a yellow exudatecontaining a high concentration of anthraquinone compounds that has beenused throughout the centuries as a cathartic and for medicinal purges;and second, a clear mucilaginous gel that has been used since ancienttimes to treat burns and other wounds where it is thought to increasethe rate of healing and reduce the risk of infection. See Grindlay, D.Reynolds, T., J. Ethnopharmcol. 1986, 16(2-3), 117-151; and Joshi, S.P., J. Med. Aromat. Plant Sci. 1998, 20(3), 768-773.

For centuries, the Aloe plant has been considered to have, and has beenused, for its medicinal and therapeutic properties without any clearunderstanding or scientific analysis of the bases for such properties.Aristotle was aware that the healing properties of Aloe would beinvaluable to soldiers wounded in battle and advised his studentAlexander III (“the Great”) to conquer all lands that grew it,especially the island of Socotra off the coast of eastern Africa.Pedanius Dioscorides, a physician in the Roman army, mentioned medicinalAloes in his encyclopedic Greek herbal De Materia Medica (approximately75 BC). Further, it is known that the biological activities of freshAloe plant decay quite rapidly.

Over that past few decades there has been a high level of interest indeveloping an understanding of the active components of Aloe that areresponsible for its beneficial effects relating to enhanced immunefunction. The research in this area has focused largely onpolysaccharides and other carbohydrate-containing substances such asglycoproteins.

Active chemical substances and mixtures, including for example,polysaccharides or other carbohydrate-containing substances of Aloeleaves have been identified, isolated and stabilized as described inU.S. Pat. No. 7,196,072, (“the '072 patent”), U.S. Pat. Nos. 5,902,796,4,735,935, 4,851,224, 4,917,890, 4,957,907, 4,959,214, and 4,966,892.

One group of the active chemical substances has been referred to as Aloevera mucilaginous polysaccharides, which are made up of a mixture ofpolysaccharides. The term “polysaccharides” has been used loosely toinclude both oligomers and polymers of carbohydrates or simple sugarsand is used in a similar manner herein. One group of suchpolysaccharides has been given the name acemannan.

According to literature, acemannan is an ordered linear polymer ofsubstantially acetylated mannose monomers available from CarringtonLaboratories, Inc. (Irving, Tex.), and has been shown in laboratorystudies to increase up to 300%, in 48 hours, the replication offibroblasts in tissue culture, which are known to be responsible forhealing burns, ulcers and other wounds of the skin and of thegastrointestinal lining.

Over a three year period, laboratory rats, the stomachs of which reactsimilarly to that of humans, were tested. Acemannan was found to beequivalent to or superior to then current medications used for thetreatment of gastric ulcers. Most such products acted to inhibithydrochloric acid in the stomach. Acemannan worked on a differentprinciple and apparently did not alter the natural flow of digestiveacids. See U.S. Pat. No. 5,902,796, column 2, lines 20-25.

Acemannan has been shown to be an inducer of IL-I and prostaglandin E2(PGE2) production by human peripheral blood adherent cells in culture.IL-I is an important macrophage product reported in the literature toinfluence the activity and production of lymphocytes, fibroblasts,B-lymphocytes and endothelial cells. See Old, Scientific American,258(5):59-60, 69-75 (1988).

IL-1 induces fibroblast proliferation which is fundamental to woundhealing. IL-1 also: (1) enhances bone marrow activity; it may betherapeutic in individuals whose bone-marrow is depressed; and (2)enhances the immune system in general.

According to literature, a series of experiments with mixed lymphocytecultures (MLC) has shown that acemannan increases the alloantigenicresponse of these lymphocytes in a dose-related fashion. Incubation ofacemannan with monocytes permitted monocyte-driven signals to enhancethe T lymphocyte response to leetin. Related studies on acemannan'seffects on MLC have shown an increase in phagocytosis and activity ofnatural killer cells. In these in vitro test systems, acemannan wasallegedly non-toxic and an immunoenhancer.

Acemannan allegedly stimulates lymphocytes to secrete lymphokines andalso causes HIV-infected lymphocytes to produce altered glycoproteins(GP-120) by a mechanism similar to that of glucosidase I inhibitors. SeeGruters et al., Nature 330:74-77(1987) and Pal et al., Intervirol.30:27-(1989). Acemannan is phagocytized and apparently pumped to theGolgi/glycoprotein apparatus of the monocyte where it interferesdirectly with glycoprotein synthesis.

Other uses of Aloe products have been described in, for example, U.S.Pat. Nos. 5,106,616, 5,118,673, 5,308,838, 5,441,943, and 5,443,830. Thedescribed studies have primarily focused on the activities of bioactivechemical substances of Aloe vera as antiviral agents, antitumor agents,immunostimulants, immunomodulators, vaccine adjuvants, means of reducingopportunistic infections, means of controlling inflammation, means ofstimulating the immune system and wound healing processes.

A number of other products based on polysaccharide-containing fractionsisolated from Aloe vera have been published in the patent literature andelsewhere. For example, a polyuronide with a molecular weight between275,000 and 374,000 daltons is reported to be useful in treatment ofsurface wounds. (See Farkas, A., U.S. Pat. No. 3,103,466 (1963).)

The 70 kD polysaccharide, Aloeferon, has also been reported to havetherapeutic potential. (See Madis, V. H.; Omar, M. M.; Madis, V., U.S.Pat. No. 4,861,761 (1989).)

Other active components isolated from Aloe include a polysaccharidebetween 420,000 and 520,000 daltons comprised of equal amounts ofglucose and mannose. (See Farkas, A., U.S. Pat. No. 3,362,951 (1968).)

In addition, several groups have enzymatically prepared alteredpolysaccharide compositions from the naturally occurring carbohydratesin Aloe (See Strickland, F. M.; Pelley, R. P.; Kripke, M. L., U.S. Pat.No. 5,824,659 (1998)).

Aloe vera polysaccharides have also been shown in controlled studies toincrease the rate of healing in animals. Aloe vera polysaccharides havealso been shown to be an effective treatment for gastric ulcers inanimal studies.

Anti-inflammatory activity of Aloe vera gel has been reported by bothoral testimonies and respected scientific journals. Rubel [Cosmetics andToiletries, 98:109-114 (1983)] discussed the possible mechanism of theanti-inflammatory effect of Aloe gel. Ukai et al., [Journal ofPharmacobio-Dynamics, 6(12):983-990 (1983)] noted anti-inflammatoryactivity of polysaccharides extracted from the fruiting bodies ofseveral fungi. The polysaccharides allegedly demonstrated a significantinhibitory effect on carrageenan-induced edema. One of the polymers,O-acetylated-D-mannan (T-2-HN), in addition demonstrated a more markedinhibitory effect than phenylbutazone on scald hyperalgesia. Ukai etal., supra. The assertion in this article, that the polysaccharide isallegedly free from protein and lipids suggests that theanti-inflammatory effect is due to the acetylated mannan only.

Other researchers have also reported anti-inflammatory effects ofcomplex polysaccharides [Saeki et al., Japanese Journal of Pharmacology,24(1):109-118 (1974)], glycoproteins [Arita et al., Journal ofBiochemistry, 76(4):861-869 (1974)] and sulfated polysaccharides [Rochaet al., Biochemical Pharmacology, 18:1285-1295 (1969)].

It is therefore possible that mucilaginous gel, a component of the Aloevera plant, which is essentially a polysaccharide, and componentstherein, are responsible in part for Aloe vera's medicinal properties.The controversy over whether the polysaccharide is a glucomannan,mannan, pectin, or of some other composition, is allegedly resolved by aseries of chemical purification steps. Yagi et al., [Planta Medica,31(1):17-20 (1977)], using a slightly modified extraction method;isolated acetylated mannan (Aloe mannan) from Aloe arborescens Millervar. natalensis. Ovodova [Khim, Prior, Soedin, 11(1):325-331 (1975)],however, earlier isolated pectin as the main component of the same Aloespecies.

A number of other pharmacology studies have been conducted on Aloe veragel in recent times. Results have included more rapid healing ofradiation burns [Rowe, J. Am. Pharm. Assoc., 29:348-350 (1940)] andaccelerated healing of wounds [Lushbaugh et al., Cancer, 6:690-698(1953)]. Thermal burns treated with Aloe vera gel heal much faster thanuntreated burns [Ashley et al., Plast. Reconstr. Surg., 20:383-396(1957), Rovatto, supra, Rodriguez-Bigas et al., J. Plast. Reconstr.Surg., 81:386-389(1988)]. The gel is useful in treating leg ulcers [ElZawahry et al., Int. J. Dermatol., 12:68-73 (1973)] and in hasteningpost surgical healing (Payne, Thesis submitted to Faculty of BaylorUniversity, Waco, Tex., MS Degree). Experimental evidence suggests thatextracts of Aloe vera have anti-infectious properties [Solar, Arch.Inst. Pasteur Madagascar, 47:9-39 (1979)] and enhance phagocytosis[Stepanova, Fiziol. Akt. Veshchestva, 9:94-97 (1977)].

During commercial processing and quality control of Aloe products,microbiological analysis is recognized as an important issue (Waller TA, et al. In Aloes: The genus Aloe; Reynolds T, Ed.; CRC Press: NY,2004; Chapter 8, pp 139-205). Microbiological organisms can originatefrom both exogenous sources such as the environment and from within theAloe plant itself. The endogenous bacterial flora are microorganismsthat naturally exist within Aloe that have evolved to grow well in thatunique environment. The primary interest in endogenous bacteria has beenwith respect to controlling its potential overgrowth that can resultwith improper post-harvest processing methods (e.g. lack of or incorrectpasteurization). Furthermore, it has been highlighted that highbacterial load in Aloe products is likely to result in loss ofbiological activity. This has been reported by the observation that highbacterial content (>100,000 organisms/gram of gel) is correlated withloss of Aloe's ability to protect the skin immune system fromUVB-induced suppression (Waller T A, et al. In Aloes: The genus Aloe;Reynolds T, Ed.; CRC Press: NY, 2004; Chapter 8, pp 139-205).

References in the art describe bacterial components in Aloe with respectto endotoxin (also referred to as lipo-polysaccharides) contamination asa confounding factor in evaluating the biological activity of purportedactive substances such acemannan polysaccharide. For example, this pointwas stressed by Tizard (Tizard I R and Ramamoorthy L. In Aloes: Thegenus Aloe; Reynolds T, Ed.; CRC Press: NY, 2004; Chapter 13, pp311-332): “It is difficult to produce Aloe carbohydrate solutions freeof contaminating endotoxin. Early studies on this material containedsmall but significant quantities of bacterial endotoxin and it ispossible that some of the biological activities ascribed to acemannanmay have been endotoxin effects”. Thus, bacterial products in Aloepreparations are considered contaminants.

U.S. Pat. No. 5,902,796 describes various methods and separationprocesses for obtaining different factors from Aloe vera. One factordescribed is called a “microparticulate factor” that can be obtained asa pellet by centrifugation at 20,000 g of the supernatant obtained froma low speed centrifugation (180 g) of an Aloe solution. Methods are alsodescribed for obtaining this factor using filtration. Thismicroparticulate factor is an activator of macrophages. The size of thismicroparticulate factor is estimated to be approximately 1 μm.

The characterization of the microparticles was described in a laterpaper (Ni Y, et al, International Immunopharmacology, 2004, 4:1745-1755). The microparticles were reported to contain galactose-richpolysaccharide(s) with the following composition: 40.2% galactose, 32.2%mannose, 20.6% glucose and other minor sugars. This paper identifies themicroparticles as degenerated cellular organelles of mesophyll cells, assummarized: “Following disruption of pulp by homogenization, threecomponents were isolated by sequential centrifugation. They were thinclear sheets, microparticles and a viscous liquid gel, which correspondto cell wall, degenerated cellular organelles and liquid content ofmesophyll cells based on morphological and chemical analysis”. Clearlythese microparticles are not believed to be bacteria but rather have adefined carbohydrate composition and are plant structural components(i.e. degenerated cellular organelles of mesophyll cells).

Gram positive micrococcus bacterial species are the most prevalentAloe-associated organisms. These bacteria have evolved to grow wellwithin the Aloe plant and do not grow very well in other environments(Waller T A, et al. In Aloes: The genus Aloe; Reynolds T, Ed.; CRCPress: NY, 2004; Chapter 8, pp 139-205).

Recent research indicates that consumption of foods containing certaingram positive bacteria can have a beneficial effect on the immunesystem. This has been most extensively studied with respect to theingestion of the probiotic lactic acid bacteria Lactobacillus andBifidobacterium strains found in yoghurt and similar foods (See, NutrRev., 2006, 64: 1-14). Many of these immune enhancing effects do notrequire viable bacterial cells since they can be mimicked by consumptionof heat killed organisms.

Problems often associated with aging include a loss of skin elasticityand muscle or joint pain. These symptoms may or may not be related andwhile significant steps are being made in the understanding of the agingprocess, much is still unknown. Treatments for skin elasticity are oftencosmetic in nature only. By covering the skin with a cosmetic, a moreyouthful appearance can be obtained in some instances. Some productsactually glue the loose skin making it appear more taught. There arealso surgical methods which can involve removing excess skin and/ortightening the skin in procedures commonly referred to as “face lifts”.If, however, skin elasticity could be improved by the use ofpreparations which are ingested or applied topically, and which actuallyeffect the elasticity of the skin directly, surgery could be postponedor avoided completely and fewer cosmetic products might be needed.

Similarly, there are many possible causes of joint pain includingarthritis and many strategies to treat them. These include topicalproducts, pharmaceutical preparations and even joint replacementsurgery. Still, as to either joint pain, or skin elasticity,improvements in cure methods are necessary. If a product whose activeingredient is all-natural could be used to provide primary relief, orcould be combined with other strategies, the results would be highlydesirable.

SUMMARY OF THE INVENTION

The present invention relates to, in one aspect, Aloe vera derivedmaterials exhibiting at least a certain amount of immunostimulatoryactivity (“IAvDs”). The degree of immunostimulatory activity required toqualify as an IAvD as measured by NF-kappa Beta directed luciferaseexpression in THP-1 monocytes at a concentration of about 250 μg/mL orless is at least about 50% of the maximal NF-kappa Beta directedluciferase expression achieved by bacterial lipo-polysaccharide at aconcentration of about 10 μg/mL. The standard for this measurement iscrude bacterial lipo-polysaccharide “ELPS” (E. coli, serotype 026:B6)obtained from Sigma Chemical Co.).

Another aspect of the invention is compositions comprising a mixture oflipo-proteins, and lipo-polysaccharides, and optionally, also at leastone polysaccharide, all of which can be isolated from Aloe vera,although the mixture can be produced synthetically. The mixtures arereferred to herein as “PLL”. IAvD and PLL mixtures may contain two ormore materials, in any combination. These mixtures may be blended withpharmaceutical carriers or excipients.

In a preferred embodiment, an immunostimulatory composition comprises amixture of at least any two of (1) aloe-derived polysaccharides, (2)aloe-derived lipo-proteins, and (3) aloe-derived lipo-polysaccharides,the mixture exhibiting an immunostimulatory activity of at least about50% of a maximal NF-kappa Beta directed luciferase expression achievedby a bacterial lipo-polysaccharide at a concentration of about 10 μg/mL,wherein the immunostimulatory activity is measured by NF-kappa Betadirected luciferase expression in THP-1 monocytes at a concentration ofabout 250 μg/mL or less.

The mixture may comprise aloe-derived polysaccharides, aloe-derivedlipo-proteins, and aloe-derived lipo-polysaccharides

In a preferred embodiment, the PLL is a particular mixture, referred toherein as “AloeEx”, comprising 1) the Aloe vera derived polysaccharidedisclosed in the '072 patent, the disclosure of which is herebyincorporated by reference, 2) Aloe-derived lipo-proteins “ALP”, and 3)Aloe-derived lipo-polysaccharides “ALPS”. Preferably, this mixtureincludes sufficient immunostimulatory activity to qualify as an IAvD asdescribed herein.

Preferably, the PLL comprises between about 0.01% and about 50% byweight of the Aloeride polysaccharide and between about 0.0001% andabout 10% ALPs and ALPSs (the total combined amount of ALPs and ALPSs)by weight. In other embodiments, the Aloeride polysaccharide comprisesbetween about 1% and about 15% by weight and between about 0.0001% andabout 10% ALPs and ALPSs by weight. In another embodiment, the Aloeridepolysaccharide is about 10-15% by weight and the ALPs and ALPSs arebetween about 0.0001% to about 10% by weight of the IAvD.

It is believed that these ALPs and ALPSs are actually frommicroorganisms associated with Aloe vera. It is also believed that ALPsand ALPSs possess independent immunostimulatory properties. These may beused in addition to, or instead of immunostimulatory Aloe-derivepolysaccharides, including for example, those of the '072 patent, toprovide various benefits in the treatment or prevention of, among otherthings, any condition previously described in the art in conjunctionwith prior Aloe derivatives, such as treatment of burns and wounds,treatment of ulcers of the skin and of the gastrointestinal tract,enhancing bone marrow activity in individuals whose bone marrow isdepressed, enhancing the immune system in general, as an antiviralagent, antitumor agent, immunostimulant, immunomodulator, vaccineadjuvant, means for reducing infections, means of controllinginflammation, means of stimulating the wound healing process, means forincreasing the rate of healing, inhibiting scald hyperalgesia, rapidhealing of radiation burns and thermal burns, treating leg ulcers,hastening post surgical healing, anti-infectious applications, enhancingphagocytosis, treatment of immunodeficiency disorders, cancer,infectious diseases or other diseases and conditions, amelioration ofsymptoms, decreasing muscle and joint pain, improving metabolicprocesses, improving the appearance, texture, firmness and/or elasticityof skin, and as a dietary supplement or nutritional aid.

In another aspect of the invention, immunostimulatory compositioncomprises aloe-derived lipo-proteins. The aloe-derived lipo-proteinsexhibit an immunostimulatory activity of at least about 50% of a maximalNF-kappa Beta directed luciferase expression achieved by a bacteriallipo-polysaccharide at a concentration of about 10 μg/mL, wherein theimmunostimulatory activity is measured by NF-kappa Beta directedluciferase expression in THP-1 monocytes at a concentration of about 250μg/mL or less.

In another aspect of the invention, the composition is a pharmaceuticalcomposition comprising one or more IAvD. In one embodiment, thecomposition is a pharmaceutical composition comprising one or more PLLin combination with pharmaceutical carriers or excipients. In apreferred embodiment, the PLL is AloeEx. These pharmaceuticalcompositions may be used for any of the purposes described immediatelyabove in connection with PLLs.

Certain IAvDs that are useful in accordance with the present inventionmay be difficult to work with and formulate. There may be problemsdispersing or dissolving particular IAvDs and producing homogenousmixtures thereof. This can be particularly problematic when formulatingdosage forms. Nonhomogenous mixtures risk an unacceptable level of dosevariation from dosage form to dosage form and therefore dose to dose.Finding suitable processing aides, solvents or excipients that arecompatible with IAvDs and do not negatively affect the immunostimulatoryproperties or stability of the IAvDs and even more preferably do notunnecessarily dilute the activity thereby requiring larger quantities ofmore expensive materials, would therefore be highly desirable.

In accordance with another aspect of the invention, various “second”materials can be used in connection with a PLL or IAvD. It has beendiscovered that combinations with “second” materials can allow for theproduction of a homogenous dispersion, emulsion, suspension or solutionand the like of a PLL or IAvD. These “second” materials may or may notbe Aloe-derived materials and indeed may or may not be IAvDs (forexample, they may not be sufficiently immunostimulatory to qualify as anIAvD as defined herein).

In one preferred embodiment, however, these second materials are derivedfrom Aloe vera. In particular, it has been found that certain highlyactive first IAvDs can be dispersed, suspended, emulsified and/orsolubilized by using a second Aloe-derived material, which, in somepreferred embodiments are second IAvDs, e.g., Aloe-derived materialsthat meet the activity requirements set forth herein for IAvDs.

In other preferred embodiments the second materials are Aloe-derivedmaterials which, if they have any immunostimulating activity at all,exhibit activity at a level below that of an IAvD. These second,non-IAvD Aloe-derived materials generally have a significantpolysaccharide content wherein about 50% or more of the polysaccharidesare of a molecular weight of about 1,500,000 daltons or less. Forexample, MagnAloe AG at 250 μg/ml exhibits less than 25% activation.

Moreover, these second materials should also improve or enhance theprocessability of the first IAvD. The amount of this second Aloe-derivedmaterial used (whether a second IAvD or a second non-IAvD) should be anamount which is sufficient to allow for the production of, as desired, ahomogenous dispersion, solution, suspension, or emulsion of a firstIAvD. Generally at least about 10% of this second Aloe-derived materialis required, by weight percent, relative to the weight percent of thefirst IAvD and the second Aloe-derived material.

In a preferred embodiment in accordance with the present invention, thefirst material is a PLL and the second material is an Aloe-derivedmaterial which is not an IAvD, has a polysaccharide content as discussedabove (generally at least 50% less than 1,500,000 daltons) and permitsthe formation of a stable, homogeneous dispersion, solution, suspension,or emulsion. In a particularly preferred embodiment, the PLL is AloeExand the second material is MagnAloe AG. Methods of making theseformulations are also contemplated.

In still another aspect of the invention, there is provided a method oftreating muscle or joint pain, for improving the appearance, texture,firmness and/or elasticity of skin and/or for providing acceleratedgrowth of new skin cells comprising administering, to an individual inneed thereof, a therapeutic dose of an IAvD or PLL composition. Thecomposition may be administered orally, topically or transdermally, onceor several times a day and may be administered alone, with apharmaceutically acceptable carrier or excipient, and/or with otheractive or inactive ingredients.

In one embodiment of this aspect, the IAvD is a PLL.

In a particular embodiment, the PLL is AloeEx.

In another embodiment, the IAvD composition comprises an Aloe derivedpolysaccharide or mixture of polysaccharides, such as, withoutlimitation, an Aloe-derived polysaccharide such as that described in the'072 patent.

Another aspect of the invention is a method for preparing an IAvDcomprising providing an Aloe vera source material; extracting a liquidAloe solution from the Aloe vera source material; filtering the liquidAloe solution; adding a flocculent, thereby creating aggregatescontaining microorganisms or microorganism components/fragments; andcollecting the aggregates.

In another embodiment, the collected aggregates can be processed toremove anthraquinones, if present.

In an embodiment, the Aloe vera source material is selected from thegroup consisting of Aloe vera gel, whole leaf Aloe vera, Aloe vera rind,Aloe vera mucilage and Aloe vera pups. Aloe vera pups are small Aloevera plants usually considered a waste product.

In a preferred embodiment, the Aloe vera source material comprises Aloevera rind and Aloe vera mucilage. Other Aloe based materials, such asspent Aloe materials typically considered as waste materials from otherprocesses may be used.

In another aspect of the invention, method for preparing a chemicallystandardized Aloe vera derived material comprises: providing an Aloevera derived material; determining the content of 2,3-dihydroxypropylcysteine in the Aloe vera derived material; and comparing the amount of2,3-dihydroxypropyl cysteine in the Aloe vera derived material to theamount of 2,3-dihydroxypropyl cysteine in a standard preparation,thereby determining a standardized value for the Aloe vera derivedmaterial.

In one embodiment, the method further comprises purifying the Aloe veraderived material prior to determining the content of 2,3-dihydroxypropylcysteine in the Aloe vera derived material.

In yet another aspect of the invention a method for preparing abiologically standardized Aloe vera derived material comprises:providing an Aloe vera derived material; determining the level ofactivation of immune cells of the Aloe vera derived material; andcomparing the level of activity of the Aloe vera derived material to astandard immunostimulatory value, thereby determining a standardizedactivity value of the Aloe vera derived material.

In one embodiment, the method further comprises purifying the Aloe veraderived material prior to determining the level of activation of immunecells of the Aloe vera derived material.

In another aspect of the invention a method of enhancing immune functioncomprises administering, to an individual in need thereof, a therapeuticdose of at least one IAvD.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the glcosyl residue composition of AloeEx Batch 56 (ALOE056) by mole % of total carbohydrate.

FIG. 2 depicts a statistically significant decrease in mean skin reboundtime in seconds (an indicator of an increase in skin elasticity orfirmness).

FIG. 3 identifies skin elasticity study demographics by gender and age.

FIG. 4 depicts study panelist demographics.

FIG. 5 identifies measurement randomization parameters.

FIG. 6 identifies treatment/group randomization.

FIGS. 7A-7D depict the statistical analysis of Cutometer measurementsfor both the test material group and the placebo group.

FIGS. 8A-8B depict questionnaire results for test subjects assigned theactive treatment.

FIGS. 9A-9B depict questionnaire results for test subjects assigned theplacebo treatment.’

FIGS. 10A-10B depict that TLR2 is involved in NF-kappa B activation byAloeEx.

FIG. 11—depicts the response of THP-1 cells to AloeEx (batch 056) andAloeEx crude fractions.

FIG. 12—depicts proteinase K digestion and SDS polyacrylamide gelanalysis of 4% SDS extract of AloeEx phenol layer fraction.

FIG. 13—depicts lipoprotein lipase digestion of 4% SDS extract of AloeExphenol layer fraction.

FIG. 14—depicts the response of THP-1 cells to different Aloe vera leafparts.

DETAILED DESCRIPTION

While the specification concludes with the claims particularly pointingand distinctly claiming the invention, it is believed that the presentinvention will be better understood from the following description.

All percentages and ratios used herein are by weight of the totalcomposition and all measurements made are at 25° C. and normal pressureunless otherwise designated.

All temperatures are in Degrees Celsius unless specified otherwise.

The present invention can comprise (open ended) or consist essentiallyof the components of the present invention as well as other ingredientsor elements described herein. As used herein, “comprising” means theelements recited, or their equivalent in structure or function, plus anyother element or elements which are not recited. The terms “having” and“including” are also to be construed as open ended unless the contextsuggests otherwise. As used herein, “consisting essentially of” meansthat the invention may include ingredients in addition to those recitedin the claim, but only if the additional ingredients do not materiallyalter the basic and novel characteristics of the claimed invention.Preferably, such additives will not be present at all or only in traceamounts. However, it may be possible to include up to about 10% byweight of materials that could materially alter the basic and novelcharacteristics of the invention as long as the utility of thecompositions (as opposed to the degree of utility) is maintained.

All ranges recited herein include the endpoints, including those thatrecite a range “between” two values. Terms such as “about,” “generally,”“substantially,” and the like are to be construed as modifying a term orvalue such that it is not an absolute, but does not read on the priorart. Such terms will be defined by the circumstances and the terms thatthey modify as those terms are understood by those of skill in the art.This includes, at very least, the degree of expected experimental error,technique error and instrument error for a given technique used tomeasure a value.

Note that while the specification and claims may refer to a tablet orother dosage form of the invention as, for example, containing particleshaving a certain particle size or distribution, or a certain type of,for example, a filler, it may be difficult to tell from the final dosageform that the recitation is satisfied. However, such a recitation may besatisfied if the materials used prior to final blending and formulation,for example, meet that recitation.

In another example, while it might be difficult to know the weight gainthat the coating contributed to a coated particle or its particle sizedistribution as it actually exists in a finished dosage form, if it isdetermined that the coated particles used to make the dosage form, priorto, in the case of a tablet, for example, a final blending andcompression step, did exhibit the desired coating level and/or particlesize, that is sufficient.

Indeed here, it might be difficult to establish the content of aparticular component in a particular formulation or the activity of aparticular component in the final formulation. If this can beestablished based on the starting or intermediate materials, it issufficient. Indeed, as to any property which cannot be ascertained fromthe finished product directly, it is sufficient if that property residesin the formulation just prior to final production. It will beappreciated that immunostimulatory activity sufficient to define an IAvDas described herein may be established any time before or afterformulation.

As used herein, treatment and/or improvement, when used in connectionwith joint pain or skin elasticity, or indeed other conditions describedherein, refers to any lessening, whether permanent or temporary, lastingor transient, of joint or muscle pain or any increase in skin elasticitythat can be attributed to or associated with administration of the PLLand/or IAvD composition. Treatment and improvement also include use ofproducts as described herein to prevent a condition or its recurrence.Note also that improvement and treatment are used interchangeably,unless the text or context dictate otherwise.

In particular, to qualify as an IAvD herein, a material mustsubstantially contribute to the immunostimulatory properties of thecomposition. As used herein, the phrase “substantially contributes” tothe immunostimulatory properties of the composition refers to animmunostimulatory effect as described herein.

As used herein, the terms immunostimulatory, immunostimulatory effect orimmunostimulatory properties and/or activity relate to the ability ofIAvD's in accordance with the invention to cause macrophage activation,and in particular, immunostimulatory activity as indicated by increasedexpression of NF-kappa Beta directed luciferase expression in THP-1monocytes as described previously (equal to about 50% that of ELPS at 10μg/mL at a concentration of 250 μg/mL or less). This may be measuredbased on any individual Aloe-derived component or collection or mixturethereof. Thus, activity can, for example, reflect that of anAloe-derived polysaccharide or its combination with ALP and/or ALPS. Amethod for measuring this type of macrophage activation, hereinincorporated by reference, is disclosed in the '072 patent, column 5,line 12 to column 6, line 22.

Generally, macrophage activation can be measured using a luciferasereporter gene assay in THP-1 human monocytic cells. This assay measuresimmunostimulatory activity as indicated by increased expression of aNF-kappa Beta-driven luciferase reporter. THP-1 cells are cultured inRPMI 1640. medium supplemented with fetal bovine serum (10% v/v) andamikacin (60 mg/L) at 37° C., under 5% CO₂ and 95% air. Actively growingcells are transiently transfected using DEAE-dextran (10 μg/l×10⁶ cells)and the pBIIXLUC reporter plasmid (1 μg/l×10⁶ cells) containing twobinding sites for NF-kappa Beta. Transfection solution containing THP-1cells are incubated for 7 minutes in 37° C. water bath. The transfectedcells are then resuspended in 10% FBS, RPMI 1640 medium and plated outin 96-well plates at a cell density of 2×10⁵ cells per well. After24-hours test samples are added to transfected cells. Four hours afteraddition of samples, cells are harvested and luciferase activitymeasured. Cells are harvested using Packard filter plates and lysedusing 30 μL of luciferase mix (1:1, LucLite™ luciferase:1×PBS, 1 mM Caand Mg). Luciferase light emission is measured using a Packardmicroplate scintillation counter in single photon mode. Activation isreported as a percentage relative to maximal activation of NF-kappa Betaby 10 μg/mL ELPS, used as a positive control. Other assay methods arecontemplated.

As used herein, the term non-immunostimulatory Aloe vera-derivedmaterial and other words indicating this concept are defined as an Aloevera-derived material which does not meet the immunostimulatoryrequirements of an IAvD as defined herein.

The term Aloe-derived lipo-protein or “ALP” refer to bacteriallipo-proteins from bacteria endogenous to Aloe vera. This does not meanthat the bacteria must come from Aloe vera as the bacteria could beseparately cultured or cultivated. Gram positive micrococcus bacterialspecies are the most prevalent Aloe-associated organisms. These bacteriahave evolved to grow well within the Aloe plant and do not grow verywell in other environments (Waller T A, et al. In Aloes: The genus Aloe;Reynolds T, Ed.; CRC Press: NY, 2004; Chapter 8, pp 139-205). Grampositive bacteria contain lipoproteins, but no lipo-polysaccharides. Asdisclosed herein, lipoproteins are believed to be a significantcontributor to in vitro macrophage activation from gram positivebacteria. Since gram positive bacteria are the most prevalent type ofbacteria in Aloe, lipoproteins represent an important active derivedfrom Aloe.

Recent research indicates that consumption of foods containing certaingram positive bacteria can have a beneficial effect on the immunesystem. This has been most extensively studied with respect to theingestion of the probiotic lactic acid bacteria Lactobacillus andBifidobacterium strains found in yoghurt and similar foods (reviewed inEzendam and van Loveren, Nutr Rev., 2006, 64: 1-14). Many of theseimmune enhancing effects do not require viable bacterial cells sincethey can be mimicked by consumption of heat killed organisms.

As used herein, the terms Aloe vera derived lipo-polysaccharide and ALPSrefer to a large molecule comprising a lipid and a polysaccharide(carbohydrate) joined by a covalent bond. ALPSs are a major component ofthe outer membrane of Gram-negative bacteria found in or associated withAloe vera, contributing greatly to the structural integrity of thebacteria, and protecting the membrane from certain kinds of chemicalattack. ALPS are endotoxins, and induce a strong response from normalanimal immune systems.

LPS generally act as the prototypical endotoxin, because it binds theCD14/TLR4/MD2 receptor complex, which promotes the secretion ofpro-inflammatory cytokines in many cell types, but especially inmacrophages. An “LPS challenge” in immunology is the exposing of thesubject to an LPS which may act as a toxin. LPS also increases thenegative charge of the cell membrane and helps stabilize the overallmembrane structure. LPS are additionally an exogenous pyrogen (externalfever-inducing composition). ALPs are different from ALPSs, also adesirable component of the PLLs of the invention, in that theirimmunostimulatory action is believed to be mediated through toll-likereceptor 2, whereas ALPS are believed to be mediated through toll-likereceptor 4.

Some IAvD compositions have no ALPs or ALPSs present that substantiallycontribute to the immunostimulatory properties of the composition. OtherIAvD compositions however, include no polysaccharides, other than ALPSsthat substantially contribute to the immunostimulatory properties of thecomposition. Still other IAvD compositions have only ALPs whichsubstantially contribute to the immunostimulatory properties of thecomposition.

Preferably, there are two or more of polysaccharides, ALPS and/or ALPSs,any or all of which substantially contribute individually orsynergistically to the immunostimulatory activity of the IAvDcomposition.

Some IAvD compositions include ALPS and/or ALPSs havingimmunostimulatory activity which are derived from microorganisms such ascertain bacteria, yeasts, fungi, molds and the like found in orotherwise associated with Aloe vera. These ALPS and ALPSs may also berecovered from microorganisms which have been cultured, so long as thatgenus and species are found on or associated with Aloe vera. These ALPsand ALPSs, which may be found on or within the Aloe vera plant, whichmay be concentrated or synthesized, may be used in addition to, orinstead of polysaccharides to provide various benefits in the treatmentof, among other things, joint and muscle pain, and enhancing theappearance, texture, firmness, and/or elasticity of skin. PLL'scontaining Aloe-derived polysaccharides, like that described in the '072patent, as well as both ALPs and APLS's are contemplated for these uses.

As used herein, the term Aloe-derived material refers to any materialcontained in or present on Aloe vera including but not limited toAloe-derived polysaccharides, lipo-proteins and lipo-polysaccharides.Such materials can be isolated from the Aloe plant and/or synthesized.

As used herein, the term synthetic or synthesized refers to materialswhich are manmade and/or manufactured and which have the same or similarcompositions of those materials found in nature. In the case of ALPs andALPSs, it also refers to separately cultured.

As used herein, the term “AloeEx” refers to an immunostimulatory PLLmixture, extracted from Aloe vera or produced synthetically, comprisingthe polysaccharide of the '072 patent as well as ALP and ALPS. A PLLsuch as AloeEX can be isolated from the rind and/or the mucilage of Aloevera leaves (i.e. the region between the inner gel and the outer rind)and may be obtained from Aloe waste materials (i.e. spent rinds used inother processes and considered a waste product by current industrystandards).

AloeEx often has an immunostimulatory activity, as measured by thetechniques described herein, of 5 μg/mL. This means that AloeEx at aconcentration of about 5 micrograms/milliliter increases NF-kappa Bdirected luciferase expression in THP-1 monocytes comparable to levels50% of those realized by the use of ELPS at a concentration of 10 μg/mL,or more. Of course, batch to batch variations can occur within thislevel of activity.

As used herein, the term Aloeride (as distinguished from the trademarkALOERIDE® discussed below) refers to an immunostimulatory polysaccharidecomposition, isolated from Aloe vera or produced synthetically, whereinthe polysaccharides have an apparent molecular weight greater than abouttwo million daltons and comprise glucose, galactose, mannose andarabinose as reported in the '072 patent. In particular, the Aloeridepolysaccharide composition may comprise between about 25% and about 70%glucose, between about 0.5% and 35% rhamnose, between about 5% and about30% galactose, between about 3% and about 25% mannose and between about0% and about 15% arabinose (in mole %). In the '072 patent the Aloeridepolysaccharide is also called NP18298. For the purpose of the currentinvention, Aloeride and NP18298 refer to the same composition.

As used herein, the term MagnAloe AG refers to a powdered mixture ofAloe-derived polysaccharides having a molecular weight distribution asdescribed at http://www.firstAloevera.com/magnAloe_information.htm. Anymixture comprising high molecular weight Aloe-derived polysaccharidematerials is contemplated for the purpose of an IAvD or non-IAvD asdescribed herein.

Preliminary findings leading to the '072 patent suggested thatdetectable levels of immunostimulation in crude Aloe juice and gel couldnot be accounted for by previously known Aloe components. CommercialAloe preparations (200 μg/mL) only resulted in negligible NF-kappa Betaactivation in the macrophage assay. Aloeride “NP18298”, which accountedfor this activity, was found to comprise a polysaccharide-containingfraction where the polysaccharides present had an apparent molecularweight above 2 million daltons. Its major glycosyl components reportedtherein included glucose, galactose, mannose and arabinose. Based on thedegree and methods of purification used and reported in the '072 patent,it is not believed that Aloeride included substantial levels of ALPsand/or ALPSs.

In investigating alternate sources and methods of scaling-up productionof Aloeride-containing materials, it was subsequently discovered thatother fractions, fractions which could include the fraction described inthe '072 patent, along with other materials including ALPs and ALPSs,possessed desirable immunostimulatory activity.

The methods described in the '072 patent, using the starting materialsdescribed, provided a relatively pure polysaccharide-containingfraction, devoid of sufficient ALPs and ALPSs to provide significantadditional immunostimulatory activity.

Without wishing to be bound by any particular theory of operation, it isbelieved that ALPs and ALPSs possess separate immunostimulatoryproperties and may be used in addition to, or instead of polysaccharidesto provide various benefits in the treatment of, among other things,joint and muscle pain, and skin elasticity enhancement.

The results of testing indicate that AloeEx which comprises the Aloeridepolysaccharide, ALPs and ALPSs, are collectively responsible for themacrophage activation properties (immunostimulatory properties) ofAloeEx.

IAvD's may include PLL's, AloeEx, mixtures of ALPs and ALPSs,polysaccharides and the Aloeride polysaccharide (which is substantiallyfree from ALPs and ALPSs). IAvD's may also include other materials,including commercial polysaccharide preparations havingimmunostimulatory derived compositions and which have been shown tocause activation of NF-kappa Beta directed luciferase expression inTHP-1 cells to the extent that they meet the criteria for IAvD's setforth herein.

As stabilized formulations and compositions of IAvD's are desirable,such formulations may include second Aloe-derived non-IAvD materials incombination with IAvD materials. Second Aloe-derived non-IAvD materialsinclude materials with lower immunostimulatory activity and at least 50%of the polysaccharides generally having a molecular weight of 1,500,000daltons or less.

If the IAvD is mixed with a second Aloe-derived material, whether asecond IAvD or non-IAvD, of course, the percentage of individualcarbohydrates, and indeed the overall molecular weight can be changed.Moreover, the overall activity of the mixture may change. For example, amixture of AloeEx and MagnAloe AG 20%:80% mixture will generally have alower overall molecular weight for the polysaccharides, will have alower overall activity than AloeEx and will have mannose as the mostprevalent carbohydrate.

Non-limiting examples of these second, non-IAvD Aloe-derived materialsinclude those selected from the group consisting of MagnAloe AG, Aloevera gel 1× (either natural, ground filet, micronized filet ordecolorized), Aloe vera gel 10× (either natural or decolorized), Aloevera gel 200× (either dehydrated powder or spray dried powder), Aloevera whole leaf spray dried powder 100×, Aloe vera whole leafdecolorized liquid (either 1×, 2×, 4×, or 10×) acemannan, Aloe veramucilaginous polysaccharide (AVMP) and Manapol.

The amount of the second Aloe-derived material present in theformulation generally is at least about 10% to about 90% by weightrelative to the amount of the total of the second Aloe-derived materialand the first IAvD. The greater the ability of the second Aloe-derivedmaterial to solubilize, suspend, emulsify and/or disperse the firstIAvD, as well as the greater the activity of the second Aloe-derivedmaterial, the less that may be needed. In one preferred embodiment, thesecond Aloe-derived material is MagnAloe AG and the amount of MagnAloeAG is greater than the amount of AloeEx, and in a particularly preferredembodiment, they are provided in a 4:1 weight ratio MagnAloe AG toAloeEx. In the resulting homogenous mixture formed by the two, MagnAloeAG would form about 80% thereof. As described and defined herein,MagnAloe AG is not believed to qualify as an IAvD as defined herein.

Other IAvD's and non-IAvD's include commercial polysaccharidepreparations such as Aloe vera mucilaginous polysaccharide (AVMP) andManapol, available from Carrington Laboratories Inc. (Irving, Tex.)having immunostimulatory derived compositions and which have been shownto cause activation of NF-kappa Beta directed luciferase expression inTHP-1 cells at 200 μg/mL. In particular, AVMP and Manapol are believedto be non-IAvD's having reported activation in the range of 2-4%relative to maximal activation of NF-kappa Beta by 10 μg/mL ELPS.

Any of the abovementioned compositions may be utilized in the presentinvention either alone or in combination.

Skin Treatment

One aspect of the present invention is a method of improving theappearance, texture, firmness, and/or elasticity of skin or treatingdecreased skin elasticity in a subject in need thereof. A subject inneed thereof is any mammal, particularly, a human, in need of enhancedor improved skin elasticity. This is accomplished by administering to asubject in need thereof an effective amount of an IAvD composition for aperiod of time sufficient to obtain improvement in the subjects' skinelasticity. Neither improvement nor treatment in accordance with thepresent invention in the context of skin elasticity requires more thansome discernable, measurable improvement therein on a subjective basis.Unless stated otherwise, this can be an improvement for any subject on asubjective basis based on, for example, visual and tactile observation.

However, often improvements are measured based on a statisticallysignificant improvement. As determined by ANOVA, tests as measured byany one of the tests described herein when the methods are utilized witha defined group of subjects to be tested in a clinical study. Forexample, the Cutometer parameter Ur/Ue describes skin elasticity as itrelates to stretching and contracting. Seventy percent of Group 1subjects showed an improvement in Cutometer parameter Ur/Ue. Relative tobaseline, this significant increase in parameter Ur/Ue is equivalent toa 29% improvement at Week 12 for subjects who ingested the inventionsupplement.

The amount of improvement necessary to achieve statistical significancewill be dictated by a number of factors including, without limitation,the number of subjects, the length of the test, the techniques used formeasuring improvement in elasticity, the condition of the subject, thetype of dosage form, the daily dose used and the like. Moreover, it isnot a requirement of the present invention that statistical significancebe achieved using each and every testing methodology, so long as it isobtained in at least one test of elasticity.

The IAvD compositions of the invention can be administered through anynormal route. However, oral and topical routes, including transdermalroutes, are preferred. Of course, the route of administration candictate much of the design of the dosage forms used to accomplish same.

The amount of IAvD composition found in a single dose will be affectedby whether or not it is topically applied or ingested and for whatpurpose it is used. Generally, however, and irrespective of dosage formselected, the amount of IAvD composition used in accordance with thepresent invention used for treating skin elasticity ranges from betweenabout 0.0025 mg to about 1 g per day. This means the amount of IAvDs,not the total amount of Aloe-derived material. It may not be possible todistinguish the individual components of the IAvD formulations. It issufficient for these purposes that the IAvDs possess these propertiesprior to being mixed with other materials.

For example, an IAvD and an Aloe-derived non-IAvD may be present or anIAvD may be present.

IAvD formulations for improved skin elasticity may be given as a singledose or as divided doses throughout the day and generally the number ofdoses given will range from between 1 to about 6 per day, morepreferably 1 to 4 per day, most preferably 1 to 2 per day. This is basedon an IAvD exhibiting activity characterized by increased NF-kappa Bdirected luciferase expression in THP-1 cells to levels about 50%, asdescribed herein, of those achieved by ELPS having a concentration ofabout 10 μg/mL when tested at a concentration of about 5 μg/mL (thatgenerally found in AloeEx). Note that there is variation in this levelof monocyte/macrophage activation batch to batch, so more or less PLLmay be required to provide an equivalent level of activity. Moreover,other IAvDs may be more or less immunostimulatory when compared toPLL's. An amount of each of these IAvDs that would give comparableactivity to PLL's may be needed.

Accordingly, the amount of the IAvD composition found in each dosageform in accordance with the present invention can range from betweenabout 0.0005 milligram to about 1 gram depending upon the daily dose,the number of doses as well as the route of administration.

Most preferably, in terms of improved skin elasticity and/or acceleratedskin cell renewal by oral administration, the amount of IAvD compositionranges from between about 0.2 milligram (mg) to about 5 gram (g), morepreferably from between about 5 mg to about 1 g, per day most preferablyabout 10 mg to about 500 mg. And when provided topically the amountranges from between about 0.0005 mg to about 2.5 mg, more preferablyfrom between about 0.0025 and about 0.25 mg, and even more preferablybetween about 0.0025 mg to about 0.025 mg. These amounts are based onthe use of AloeEx at an activity of about 5 micrograms/milliliter(μg/mL). Amounts of other IAvD's which yield similar results arecontemplated.

In a preferred embodiment in accordance with the present invention, thedaily dosing of IAvD compositions continue for at least 7 days, morepreferably at least 2 weeks, more preferably at least 2 months. Mostpreferably, the materials of the present invention are administeredcontinuously throughout a subject's life to improve and maintain skinelasticity. However, the amount administered for treatment may,eventually, be reduced to accomplish maintenance of a certain desiredlevel of skin elasticity or accelerated skin cell renewal. Thus, aftersome period of treatment, once skin elasticity has improved, a lowerdose may be administered for maintenance thereof.

When applied topically, the dosage may be spread directly onto anafflicted area. It may in some cases be rubbed into the skin. In otherinstances, it is not manipulated but is allowed to be absorbed or toremain on the application area as applied. The IAvD may also be appliedon occlusive bandage or some other type of wrap placed over it.

Alternatively, the IAvD may be formulated into a patch. These topicalapplications may be designed to deliver the IAvD to the surface of theskin or to some layer or layers of the skin itself. Alternatively, itmay be formulated to deliver the IAvD transdermally for deeper local orsystemic distribution. More about the types of materials that can beused to formulate oral and topical dosage forms are discussed herein.

When administered orally, IAvD compositions may be provided in a tablet,capsule, powder, sachet, lozenge, gum candy or any other ingestibleform. The IAvD may also be provided as a liquid or as a solid which isfirst dissolved in or mixed with a liquid before ingestion. Dosage formsmay be swallowable or may be solids in the mouth and disintegrate in themouth prior to ingestion. The IAvD may even be sprinkled on food andingested therewith.

Note that “accelerated cell regeneration” is an end point in its ownright and methods of enhancing accelerated cell regeneration andproducts which accomplish this using IAvD compositions are alsocontemplated. Accelerated cell regeneration is the regeneration of skincells under dead layers of skin which are desirably exfoliated. This isaccomplished using the same techniques and products as described inconnection with skin elasticity.

Joint/Muscle Pain

Another aspect of the present invention is the improvement or thetreatment of joint and/or muscle pain in subjects in need thereof by theuse of IAvD compositions in accordance with the present invention.Generally, the routes of administration and dosage forms previouslydiscussed in accordance with skin elasticity apply equally well to thesemethods. Topical application can affect a particular muscle or musclegroup and/or a particular joint. Systemic administration (orally ortransdermally) may reduce pain at that same location and/or may providebroader relief over many muscle groups and/or joints.

Also as noted previously in connection with skin elasticity, treatmentin accordance with this aspect of the present invention means areduction in joint pain and/or muscle pain which can be measuredsubjectively on a subject-by-subject basis. However, more preferably, asuccessful product in accordance with the present invention will provideadequate treatment in a statistically significant way when given to agroup of subjects treated over an extended period of time. Desirably, atleast 75% of subjects will manifest a lower frequency of muscle and/orjoint pain, a reduction in pain intensity or a reduction in painduration after twelve weeks of treatment. This can be pain generally orin a specific muscle or joint.

Improvement in a subject's condition in the context of joint pain and/ormuscle pain is often determined by a subjective rating system scored byeach individual rating his or her pain at a specific site or generallybefore, during and/or after treatment.

Also as previously noted, the amount of IAvD composition administered inaccordance with this aspect of the present invention will vary dependingupon the route of administration, the area to be treated, the degree andtype of pain and the like. However, the amount generally will be in thesame ranges as previously described in connection with the methods ofimproving skin elasticity. It is also possible in both this method andmethods of improving skin elasticity to administer the IAvD compositionboth orally and topically at the same time or within the same day, or onsome alternative or complementary schedule thereby providing bothgeneral and more specifically targeted relief or improvement.

Any IAvD composition may be used in accordance with the presentinvention. For example, any PLL, any AloeEx or any Aloeridepolysaccharide formulation exhibiting immunostimulatory properties asdescribed herein may be used. The present invention includespharmaceutical compositions and methods of using an IAvD, optionally incombination with acceptable pharmaceutical carriers or excipients.

Compositions

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. More specifically, atherapeutically effective amount means an amount effective to preventdevelopment of or to alleviate the existing symptoms of the subjectbeing treated. Determination of the effective amounts is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

The amount of the composition administered will be dependent upon thecondition being treated, the subject being treated, on the subject'sweight, the severity of the affliction, the manner of administration andthe judgment of the individual's physician or of the individual.

As discussed above, suitable routes of administration may, for example,include oral, transmucosal, topical or transdermal.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making,powderizing, levigating, emulsifying, encapsulating, entrapping orlyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the compositions andcompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen.

For oral administration, the compositions can be administered alone orformulated readily by combining the active compositions withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the compositions of the invention to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions and the like, for oral ingestion by a patient to be treated.

Pharmaceutical preparations for oral and/or topical use can be obtainedusing a solid excipient, optionally grinding or milling the individualcomponents of a preparation and/or a resulting mixture, and processingthe components and/or mixture of granules, after adding suitableauxiliaries, if desired, to obtain for example, creams, lotions, serums,powders, effervescent formulations, tablets or dragee cores. Granulationis, however, but one of numerous known techniques which may be used.

Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP).

If desired, disintegrating agents may be added, such as PVP, sodiumstarch glycolate, cross-linked polyvinyl pyrrolidone, agar, or alginicacide or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active composition doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compositions may be dissolved or suspended insuitable liquids, such as fatty oils, liquid paraffin, or liquidpolyethylene glycols. In addition, stabilizers may be added. Allformulations for oral administration should be in dosages suitable forsuch administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in a conventional manner.

For topical administration, emulsions, microemulsions, or other topicalcreams, lotions, milks, washes, ointments, serums or gels may also beprepared. Direct emulsification of AloeEx may be possible, for examplepreparation of an AloeEx oil-in-water emulsion wherein the hydrophobiccore or an oil phase is largely composed of AloeEx. Other surfactants orformulary excipients may be added to physically stabilize the emulsion.

Pharmaceutical preparations suitable for topical administration areespecially creams, ointments and gels and also pastes, foams, tincturesand solutions that contain from approximately 0.05 to approximately 5%IAvD composition by weight. Preferably the preparations are about 0.5%IAvD composition by weight. These compositions include the total amountof Aloe-derived materials including, as appropriate, at least one IAvDand any other Aloe based materials. In the formulations discussed in theclinical studies, for example the 0.5% IAvD composition was made up of a1:4 mixture of AloeEx and MagnAloe AG.

Creams or lotions may be oil-in-water emulsions that typically containmore than 50% water. As oily base there are used especially fattyalcohols, especially those containing from 12 to 18 carbon atoms, forexample lauryl, cetyl or stearyl alcohol, fatty acids, especially thosecontaining from 10 to 18 carbon atoms, for example palmitic or stearicacid, liquid to solid waxes, for example isopropyl myristate, wool waxor beeswax, and/or hydrocarbons, especially liquid, semi-solid or solidsubstances or mixtures thereof, for example petroleum jelly (petrolatum)or paraffin oil. Suitable emulsifiers are surface-active substanceshaving predominantly hydrophilic properties, such as correspondingnon-ionic emulsifiers, for example fatty acid esters of polyalcohols orethylene oxide adducts thereof, especially corresponding fatty acidesters with (poly)ethylene glycol, (poly)propylene glycol or sorbitol,the fatty acid moiety containing especially from 10 to 18 carbon atoms,especially partial glycerol fatty acid esters or partial fatty acidesters of polyhydroxyethylene sorbitan, such as polyglycerol fatty acidesters or polyoxyethylene sorbitan fatty acid esters (Tweens), and alsopolyoxyethylene fatty alcohol ethers or fatty acid esters, the fattyalcohol moiety containing especially from 12 to 18 carbon atoms and thefatty acid moiety especially from 10 to 18 carbon atoms, especiallythose having approximately from 2 to 23 ethylene glycol or ethyleneoxide units, such as polyhydroxyethylenecetylstearyl ether (for exampleCetomacrogol), polyhydroxyethylene-(4)-lauryl ether andpolyhydroxyethyleneglycerol fatty acid ester (for example Tagat S), orcorresponding ionic emulsifiers, such as alkali metal salts of fattyalcohol sulfates, especially having from 12 to 18 carbon atoms in thefatty alcohol moiety, for example sodium lauryl sulfate, sodium cetylsulfate or sodium stearyl sulfate, which are usually used in thepresence of fatty alcohols, for example cetyl alcohol or stearylalcohol.

Additives to the aqueous phase are agents that prevent the creams fromdrying out, for example humectants, such as polyalcohols, such asglycerol, sorbitol, propylene glycol and/or polyethylene glycols, andalso preservatives, perfumes, etc.

Ointments or lotions may be water-in-oil emulsions that typicallycontain up to 70%, but preferably from approximately 20% toapproximately 50%, water or aqueous phase. Suitable as fatty phase areespecially hydrocarbons, for example petroleum jelly, paraffin oiland/or hard paraffins, which, in order to improve the water-bindingcapacity, preferably contain suitable hydroxy compounds, such as fattyalcohols or esters thereof, for example cetyl alcohol or wool waxalcohols, or wool wax.

Emulsifiers are corresponding lipophilic substances, such as sorbitanfatty acid esters (Spans), for example sorbitan oleate and/or sorbitanisostearate. Additives to the aqueous phase are, inter alia, humectants,such as polyalcohols, for example glycerol, propylene glycol, sorbitoland/or polyethylene glycol, and also preservatives, perfumes, etc.

Microemulsions are isotropic systems based on the following fourcomponents: water, an emulsifier, for example of the type indicatedabove, such as a surfactant, for example emulgin, a lipid, such as anon-polar oil, for example paraffin oil, and an alcohol containing alipophilic group, for example 2-octyldodecanol. If desired, otheradditives may be added to the microemulsions.

Fatty ointments are water-free and contain as base especiallyhydrocarbons, for example paraffin, petroleum jelly and/or liquidparaffins, also natural or partially synthetic fat, such as fatty acidesters of glycerol, for example coconut fatty acid triglyceride, orpreferably hardened oils, for example hydrogenated groundnut oil orcastor oil, also fatty acid partial esters of glycerol, for exampleglycerol mono- and di-stearate, and also, for example, the fattyalcohols increasing the water-absorption capacity, emulsifiers and/oradditives mentioned in connection with the ointments.

With gels, a distinction is made between aqueous gels, water-free gelsand gels having a low water content, which gels consist of swellable,gel-forming materials. There are used especially transparent hydrogelsbased on inorganic or organic macromolecules. High molecular weightinorganic components having gel-forming properties are predominantlywater-containing silicates, such as aluminium silicates, for examplebentonite, magnesium aluminium silicates, for example Veegum, orcolloidal silicic acid, for example Aerosil. As high molecular weightorganic substances there are used, for example, natural, semi-syntheticor synthetic macromolecules. Natural and semi-synthetic polymers arederived, for example, from polysaccharides containing a great variety ofcarbohydrate components, such as celluloses, starches, tragacanth, gumarabic and agar-agar, and gelatin, alginic acid and salts thereof, forexample sodium alginate, and derivatives thereof, such as loweralkylcelluloses, for example methyl- or ethyl-cellulose, carboxy- orhydroxy-lower alkylcelluloses, for example carboxymethyl- orhydroxyethyl-cellulose. The components of synthetic gel-formingmacromolecules are, for example, suitably substituted unsaturatedaliphatic compounds such as vinyl alcohol, vinylpyrrolidine, acrylic ormethacrylic acid. Examples of such polymers are polyvinyl alcoholderivatives, such as polyviol, polyvinylpyrrolidines, such as collidone,polyacrylates and polymethacrylates, especially having a molecularweight of from approximately 80,000 to approximately 1 million, or saltsthereof, such as Rohagit S, Eudispert or Carbopol. Customary additives,such as preservatives, stabilizers, or perfumes, may be added to thegels.

Foams are administered, for example, from pressurized containers and areliquid oil-in-water emulsions in aerosol form; unsubstituted orhalogenated hydrocarbons, such as alkanes, for example propane orbutane, or chlorofluoro-lower alkanes, for exampledichlorodifluoromethane and dichlorotetrafluoroethane, are used aspropellant. As oil phase there are used, inter alia, hydrocarbons, forexample paraffin oil, fatty alcohols, for example cetyl alcohol, fattyacid esters, for example isopropyl myristate, and/or other waxes. Asemulsifiers there are used, inter alia, mixtures of emulsifiers havingpredominantly hydrophilic properties, such as polyoxyethylene sorbitanfatty acid esters, and emulsifiers having predominantly lipophilicproperties, such as sorbitan fatty acid esters. The customary additives,such as preservatives, etc., are also added.

Tinctures and solutions generally have an ethanolic base, to which watermay be added and to which there are added, inter alia, polyalcohols, forexample glycerol, glycols and/or polyethylene glycol, as humectants forreducing evaporation, and fat-restoring substances, such as fatty acidesters with low molecular weight polyethylene glycols, that is to saylipophilic substances that are soluble in the aqueous mixture, as areplacement for the fatty substances removed from the skin by theethanol, and, if necessary, other adjuncts and additives. Suitabletinctures or solutions may also be applied in spray form by means ofsuitable devices.

The manufacture of the topically administrable pharmaceuticalpreparations is effected in a manner known per se, for example bydissolving or suspending the active ingredient in the base or, ifnecessary, in a portion thereof. When the active ingredient isadministered in the form of a solution, it is generally dissolved in oneof the two phases before emulsification; when the active ingredient isadministered in the form of a suspension, it is mixed with a portion ofthe base after emulsification and then added to the remainder of theformulation.

An IAvD composition or combination of IAvD compositions may by appliedas a topical medication for local effect, such as enhanced skinelasticity and/or accelerated skin cell growth or regeneration. This isa treatment which is applied to the skin and acts directly at the siteof application. It must be placed at the site of pathology, and itdirectly absorbs through the skin and exerts its effect. It is believedthat there is no systemic absorption needed for the effect to occur.

An IAvD composition or combination of IAvD compositions may be appliedas a topical medication for systemic effect. This is a treatment whichis applied to the skin and acts at a site distant from the site ofapplication to reduce muscle or joint pain, enhance skin elasticity orboth. The medication may be applied anywhere on the body and achieve itseffect. The formulation must absorb through the skin, enter thebloodstream, reach effective levels in the blood, and then travel to itssite of action to exert its effect.

It will be known to persons of ordinary skill in the art that skinpenetration enhancers may be used to facilitate systemic delivery of theIAvD composition or compositions or even deeper muscle or jointpenetration. In this regard, penetration enhancers have been used toincrease the permeability of the dermal surface to drugs, and are oftenproton accepting solvents such as dimethyl sulfoxide (DMSO) anddimethylacetamide. Other penetration enhancers that have been studiedand reported as effective include 2-pyrrolidine, N,N-diethyl-m-toluamide(Deet), 1-dodecal-azacycloheptane-2-one N,N-dimethylformamide,N-methyl-2-pyrrolidine, calcium thioglycolate, hexanol, fatty acids andesters, pyrrolidone derivatives, derivatives of 1,3-dioxanes and1,3-dioxolanes, 1-N-dodecyl-2-pyrrolidone-5-carboxylic acid,2-pentyl-2-oxo-pyrrolidineacetic acid,2-dodecyl-2-oxo-1-pyrrolidineacetic acid,1-azacycloheptan-2-one-2-dodecylacetic acid, and aminoalcoholderivatives, including derivatives of 1,3-dioxanes, among many others.

An IAvD composition or combination of IAvD compositions may be includedin dosage forms used to treat any responsive condition, such as, jointpain, muscle pain, loss of skin elasticity or skin wrinkles.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude but are not limited to calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Stable compositions for treatment, prevention, or amelioration of any ofthe conditions, diseases, afflictions, wounds and the like, including,without limitation one or more symptoms of joint or muscle pain and forenhancing skin elasticity are provided. The compositions provided hereinare stable formulations of an immunostimulatory Aloe vera extract or aderivative thereof, in a pharmacologically suitable fluid that arestable during long term storage. The compositions are may be suitablefor direct administration to a subject in need thereof or may be in aconcentrated form. Pharmacologically suitable fluids include, but arenot limited to, polar fluids, including protic fluids. In certainembodiments herein, the compositions are aqueous solutions. In otherembodiments, the compositions are non aqueous or emulsions.

The IAvD compositions may be formulated with a pharmacologicallysuitable fluid or solvent. Pharmacologically suitable fluids include,but are not limited to, polar solvents, including, but not limited to,compounds that contain hydroxyl groups or other polar groups. Suchsolvents include, but are not limited to, water or alcohols, such asethanol, isopropanol, and glycols including propylene glycol,polyethylene glycol, polypropyleneglycol, glycol ether, glycerol andpolyoxyethylene alcohols.

Polar solvents also include protic solvents, including, but not limitedto, water, aqueous saline solutions with one or more pharmaceuticallyacceptable salt(s), alcohols, glycols or a mixture thereof. For a salinesolution as the solvent or as a component thereof, particularly suitablesalts are those which display no or only negligible pharmacologicalactivity after administration.

The compositions provided herein also may include excipients andadditives. The particular excipient or additive for use in thecompositions for long term storage provided herein may be determinedempirically using methods well known to those of skill in the art.Excipients and additives are any pharmacologically suitable andtherapeutically useful substance which is not an active substance.Excipients and additives generally have no pharmacological activity, orat least no undesirable pharmacological activity. The excipients andadditives include, but are not limited to, surfactants, stabilizers,complexing agents, antioxidants, or preservatives which prolong theduration of use of the finished pharmaceutical formulation, flavorings,vitamins, or other additives known in the art.

Complexing agents include, but are not limited to,ethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as thedisodium salt, citric acid, nitrilotriacetic acid and the salts thereof.In one embodiment, the complexing agent is EDTA. Preservatives include,but are not limited to, those that protect the solution fromcontamination with pathogenic particles, including benzalkonium chlorideor benzoic acid, or benzoates such as sodium benzoate. Antioxidantsinclude, but are not limited to, vitamins, provitamins, ascorbic acid,vitamin E or salts or esters thereof.

The compositions provided herein also may include a cosolvent, whichincreases the solubility of additives or the active ingredient(s). Theparticular cosolvent for use in the compositions for long term storageprovided herein may be determined empirically using methods well knownto those of skill in the art. Cosolvents for use herein include, but arenot limited to, hydroxylated solvents or other polar solvents, such asalcohols such as isopropyl alcohol, glycols such as propylene glycol,polyethylene glycol, polypropylene glycol, glycol ether, glycerol, andpolyoxyethylene alcohols.

In other of embodiments, the compositions further contain a buffer,including, but not limited to, citric acid/phosphate, acetate, barbital,borate, Britton-Robinson, cacodylate, citrate, collidine, formate,maleate, McIlvaine, phFosphate, Prideaux-Ward, succinate,citrate-phosphate-borate (Teorell-Stanhagen), veronal acetate, MES(2-(N-morpholino)ethanesulfonic acid), BIS-TRIS(bis(2-hydroxyethyl)imino-tris-(hydroxymethyl)methane), ADA(N-(2-acetamido)-2-iminodiacetic acid), ACES(N-(carbamoylmethyl)-2-aminoethanesulfonic acid), PIPES(piperazine-N,N′-bis(2-ethanesulfonic acid)), MOPSO(3-(N-morpholino)-2-hydroxypropanesulfonic acid), BISTRIS PROPANE(1,3-bis(tris(hydroxymethyl)methylamino)propane), BES (N,N-bis(2-MOPShydroxyethyl)-2-aminoethanesulfonic acid),(3-(N-morpholino)propanesulfonic acid), TES(N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid), HEPES(N-(2-hydroxyethyl)piperazine-N′-(2-eth-anesulfonic acid), DIPSO(3-(N,N-bis(2-hydroxyethyl)amino)-2-hydroxypropanesulfonic acid), MOBS(4-(N-morpholino)-butanesulfonic acid), TAPSO(3-(N-tris(hydroxymethyl)methylamino)-2-hydroxypropanesulfonic acid),TRIZMA® (tris(hydroxymethylaminomethane), HEPPSO(N-(2-hydroxyethyl)piperazine-N′-(2-hydroxypropanesulfonic acid), POPSO(piperazine-N,N′-bis(2-hydroxypropanesulfonic acid)), TEA(triethanolamine), EPPS (N-(2-hydroxyethyl)piperazine-N′-acid),(3-propanesulfon-ic acid), TRICINE(N-tris(hydroxy-methyl)methylglycine), GLY-GLY (glycylglycine), BICINE(N,N-bis(2-hydroxyethyl)glycine), HEPBS(N-(2-hydroxyethyl)piperazine-N′-(4-butanesulfonic acid)), TAPS(N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid), AMPD(2-amino-2-methyl-1,3-propanediol), and/or any other buffers known tothose of skill in the art.

The particular buffer and buffer concentration of a given compositionprovided herein may be determined empirically using standard stabilityassays well known to those of skill in the art.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. Compositions comprisinga composition of the invention formulated in a compatible pharmaceuticalcarrier may also be prepared, placed in an appropriate container, andlabeled for treatment of an indicated condition. Suitable conditionsindicated on the label may include treatment of a condition.

The formulation may also contain optional ingredients including, forexample, herbs, vitamins, minerals, enhancers, colorants, sweeteners,flavorants, inert ingredients, and the like. Such optional ingredientsmay be either naturally occurring or concentrated forms. Selection ofone or several of these ingredients is a matter of formulation, design,consumer preference and end-user.

In accordance with another aspect of the present invention, variousmaterials have been found which can be used to allow for the productionof a homogenous dispersion, emulsion, suspension, solution and the likeof an IAvD. In particular, it has been found that certain highly active,but generally insoluble IAvDs can be dispersed, suspended, emulsifiedand/or solubilized by using a second Aloe-derived material. That secondAloe-derived material may be a second IAvD or a material which hasinsufficient activity to qualify as an IAvD. The amount of the secondAloe-derived material should be an amount which is sufficient to allowfor, as desired, the production of a homogenous dispersion, solution,homogeneous suspension, or homogeneous emulsion of the first IAvD.Generally at least about 10% of the second Aloe-derived material isrequired, by weight percent, relative to the weight percent of the totalweight of the first IAvD and the second Aloe-derived material.

In a particularly preferred embodiment in accordance with the presentinvention, the first IAvD is AloeEx. Any Aloe-derived material that canimprove the processability of the first IAvD may be used. The secondAloe-derived material generally is composed of polysaccharides in whichat least 50% of the polysaccharides have molecular weights of 1,500,000daltons or less. This second Aloe-derived material, can include, withoutlimitation, an Aloe based material selected from the group consisting ofMagnAloe AG, Aloe vera gel 1× (either natural, ground filet, micronizedfilet or decolorized), Aloe vera gel 10× (either natural ordecolorized), Aloe vera gel 200× (either dehydrated powder or spraydried powder), Aloe vera whole leaf spray dried powder 100×, Aloe verawhole leaf decolorized liquid (either 1×, 2×, 4×, or 10×), acemannan,Aloe vera mucilaginous polysaccharide (AVMP) and Manapol. MagnAloe AG isa powder available from First Aloe of Costa Rica, S.A. Information canbe obtained at www.firstAloevera.com/magnAloe_information.htm. MagnAloeAG has polysaccharide levels of over 800 kiladaltons (KDa) as indicatedbelow. This is on average 40% higher than spray dried 200-1 Aloe verapowder sold on the market. MagnAloe AG is reported to contain 42.65 mg/gof polysaccharides having a molecular weight (MW) of 50-200 KDa, 118.5mg/g having a MW of 200-800 KDa, 366.2 mg/g having a MW 800-1400 KDa,155.8 mg/g having a MW 1400-2000 KDa of a total polysaccharide of 683.1mg/g. The water solubility of the MagnAloe generally is about 15%.

Aloe vera gel 1× (either natural, ground filet, micronized filet ordecolorized), Aloe vera gel 10× (either natural or decolorized), Aloevera gel 200× (either dehydrated powder or spray dried powder), Aloevera whole leaf spray dried powder 100×, Aloe vera whole leafdecolorized liquid (either 1×, 2×, 4×, or 10×) are available for ImproveUSA, Inc., 215 Dalton Drive, Suite D, DeSoto, Tex. 75115. The watersolubility of Aloe vera gel 200× generally is about 90-100%.

Acemannan, Aloe vera mucilaginous polysaccharide (AVMP) and Manapol areavailable from Carrington Laboratories, Irving, Tex. In one particularlypreferred embodiment, the single most prevalent carbohydrate in the IAvDis glucose, and the single most prevalent carbohydrate in the secondAloe-derived material is mannose.

In one embodiment, the amount of the first IAvD ranges from about 10% toabout 90% by weight based on the total amount of the first IAvD andsecond Aloe-derived material and the amount of the second materialranges from about 90% to about 10% based on the amount of the first IAvDand second Aloe-derived material. In one preferred embodiment, theamount of the second material is greater than the amount of the firstIAvD, and in a particularly preferred embodiment, they are provided in a4:1 weight ratio second material to first IAvD. In the resultinghomogenous mixture formed by the two, the second material would formabout 80% thereof. In still another preferred aspect, the first IAvD isAloeEx and the second material is an Aloe-derived material that includespolysaccharides in which at least 50% of the polysaccharide havemolecular weights of about 1,500,000 daltons or less, such as MagnAloeAG and the amount of the AloeEx ranges from about 10% to about 90% byweight based on the amount of the AloeEx and MagnAloe and the amount ofthe MagnAloe AG ranges from about 90% to about 10% based on the amountof the AloeEx and MagnAloe AG. In one preferred embodiment, the amountof the MagnAloe AG is greater than the amount of the AloeEx, and in aparticularly preferred embodiment, they are provided in a 4:1 weightratio MagnAloe AG to AloeEx. In the resulting homogenous mixture formedby the two, the MagnAloe AG would form about 80% thereof.

In one embodiment, AloeEx and a second material, preferably a secondAloe-derived material may be formulated by mixing raw AloeEx with waterwhich has been heated to approximately 80° C. The mixture is agitatedfor at least 10 minutes to homogenize the material. The mixture is thenfiltered, preferably with a filter having a pore size no larger than 15microns. The resulting filtrate may be utilized in formulating a varietyof IAvD compositions as described herein.

Homogenization of AloeEx may or may not require heat depending on theequipment utilized. For example homogenizers may be used as part of theprocess to render a uniform particulate suspension. A commonly known“color” homogenizer may be used to reduce AloeEx particle size toformulate an aqueous AloeEx suspension and/or emulsion.

A characterization of AloeEx is shown in FIG. 1. The material forms abrown to green flaked powder.

Referring to FIG. 1, a chart depicts one lot of AloeEx material (batch56) with associated sugar content based on the mole % of totalcarbohydrates.

FIG. 1 shows the results of glycosyl composition analysis performed byThe University of Georgia, Complex Carbohydrate Research Center bycombined gas chromatography/mass spectrometry (GC/MS) of theper-O-trimethylsilyl (TMS) derivatives of the monosaccharide methylglycosides produced from the sample by acidic methanolysis.

Methyl glycosides were first prepared from a dry sample provided by theinventors by methanolysis in 1 M HCl in methanol at 80° C. (18-22hours), followed by re-N-acetylation with pyridine and acetic anhydridein methanol (for detection of amino sugars). The samples were thenper-O-trimethylsilylated by treatment with Tri-Sil (Pierce) at 80° C.(0.5 hours). [These procedures were carried out as previously describedin Merkle and Poppe (1994) Methods Enzymol. 230:1-15; York, et al.(1985) Methods Enzymol. 118:3-40.] A significant level of precipitatewas noted immediately upon addition of Tri Sil. GC/MS analysis of theTMS methyl glycosides was performed on an HP 5890 GC interfaced to a5970 MSD, using an All Tech EC-1 fused silica capillary column (30m×0.25 mm ID).

The monosaccharides were identified by their retention times incomparison to standards, and the carbohydrate character of these wereauthenticated by their mass spectra. For interpreting the mass spectraldata, fragment ion 73 is the characteristic base fragment for all TMSmethyl glycosides, 204 and 217 are characteristic of neutral sugars, and173 is characteristic of amino sugars. Fragment 217 is alsocharacteristic of uronic acids and fragment ion 298 is characteristic ofthe sialic acids.

FIG. 1 identifies the glcosyl residue composition by mole %. Values areexpressed as mole percent of total carbohydrate. The total percentcarbohydrate was calculated to be 8.6%. The constituent values measuredwere glucose 61.1%, arabinose 9.5%, galactose 9.2%, galacturonic acid7.9%, mannose 6.6%, xylose 1.6%, ribose 1.5%, rhamnose 1.4% and fucose1.1%.

Clinical Study—Skin Elasticity—Topical Formulation

One clinical study, as outlined below demonstrates the effect of oneIAvD composition, sold under the trademark ALOERIDE®. ALOERIDE® may beadministered topically to improve skin elasticity and may also be usedas an ingredient in a nutritional supplement.

The effect of an ALOERIDE® gel 0.5% Product #10849LL, Lot #11939 on skinelasticity or firmness, was determined in a clinical study by ConsumerProduct Testing, Inc. of Fairfield, N.J. The test formulation (ALOERIDE®gel 0.5%) comprised a 4:1 mixture by weight of MagnAloe AG and AloeExand was mixed with water, alcohol (10%) and a small amount of Carbopol(0.5%) then dispersed using a homogenizer. The dispersion of AloeEx,water, alcohol and a small amount of carbopol was used to make up thetest gel suspension.

The test product was supplied in small containers sufficient to last for8 weeks when applied to defined facial areas (alcohol performs as apreservative).

The clinical trial was a double test of AloeEx function: Carbopol tendsto be drying, a negative test performance factor estimated at (−) 15%.The clinical trial proved the ability of AloeEx/MagnAloe AG to: 1)Reduce fine lines and wrinkles in a statistical majority of the testsubjects at 4 weeks and at 8 weeks; 2) The ability of AloeEx to overcomeany negative dryness associated with carbopol.

AloeEx used in this clinical trial was non-dispersible/insoluble anddifficult to homogenize. When mixed 1:4 with MagnAloe AG, however, ahomogenous gel/emulsion was realized.

Thirty-one qualified male and female subjects, ranging in age from 40 to65 years, were selected for study. The test material indicated asignificant reduction in skin rebound time at both the 4 week and 8 weekevaluation intervals. Study participants experience up to a 72%improvement in skin elasticity or firmness at the conclusion of thestudy (FIG. 2) and 100% of the study participants experienced generalimprovement in skin elasticity. Study demographics based on age andgender are shown in FIG. 3.

Subjects reported to the laboratory with cleansed faces of the first dayof testing for a base measurement. Prior to conducting any readings orscoring, subject equilibrated in an environmentally controlled room for15 minutes at ambient conditions; temperature at 21° C.+/−2° C.;relative humidity at 40%+/−2%.

Skin elasticity (firmness) was measured using the pinch test asdisclosed by Shanahan. R. W., et al, Parameters for Assessing theEfficacy of Skin Care Products. Drug & Cosmetic Industry, 140, No. 1,42-48, 1987. Skin elasticity or firmness scores were obtained by anexpert tester by grasping the skin of the lateral, suborbital area ofthe eyes between thumb and forefinger. The resultant folds were releasedand timing of all visible movement of the skin, from release until nofurther motion, was observed and measured to the nearest one hundredthof a second. Scores were recorded on a Data Recording form forstatistical analysis of the measurements.

Improvements in skin elasticity are considered substantiated if the is astatistically significant (P less than or equal to 0.05 using thedependent t-Test) decrease in the mean score between the baselinemeasurements and the subsequent 4 week and 8 weeks post treatmentscoring intervals.

FIG. 2 shows a statistically significant decrease in mean skin reboundtime in seconds (an indicator of an increase in skin elasticity orfirmness). The mean of all baseline measurements was 2.09 seconds and1.96 seconds for left and right measurements respectively. A significantdecrease was observed at the week 4 test interval yielding 1.60 secondsand 1.53 seconds respectively. Further decreases in mean skin reboundtime to 1.19 seconds and 1.18 seconds, for left and right, were measuredat the week 8 test interval. As previously discussed, the results werestatistically significant as p was calculated to be less than or equalto zero for all measurement intervals (FIG. 2).

Another clinical study, as outlined below demonstrates the effect ofanother IAvD composition administered orally to improve muscle and/orjoint pain and skin elasticity.

Clinical Study—Skin Elasticity—Joint/Muscle Pain—Oral Formulation

A study was conducted by Clinical Research Laboratories, Inc. toevaluate the efficacy of an oral supplement of the instant inventiondesigned to improve skin firmness and elasticity and to diminish muscleand joint pain during a 12-week period. (A Double-Blind PlaceboControlled Pilot Study to Assess the Safety and Efficacy of an OralSupplement Designed to Diminish Aging, study number CRL34706 hereinincorporated by reference in its entirety). Placebo tablets used in thisclinical study contained microcrystalline cellulose, croscarmellosesodium, stearic acid, silicon dioxide, and magnesium stearate. Theactive tablets contained the same inactive ingredients as the placebotablets with 50 mg of a 4:1 mixture of MagnAloe AG and AloeEx. Thesetablets were given twice daily, orally, at the time of arising and onceat bedtime.

This study was initiated on Aug. 30, 2006 and was completed on Dec. 15,2006. Initially, safety screening was performed as follows on eachsubject.

Safety Evaluations Blood Analysis

Blood was drawn from each subject at designated study visits, and thefollowing blood tests were performed:

Complete Blood Count (CBC) with differential and platelet count.

Chemistry Panel+HDL AST (GOT), ALT (GPT), gamma Gt, LDH (LD), TotalProtein (TP), Uric Acid, (UA, total Billirubin, Urea Nitrogen (UN),total cholesterol (HDL, LDL, TG), Na, K, Ca, Cl.

Urine Analysis

A urine specimen was obtained from each subject at designated studyvisits, and the following urine tests were performed:

Glucose, Protein, Uribilinogen, Blood, pH

Sitting Vital Signs

An assessment of sitting vital signs (i.e., pulse, blood pressure andrespirations) was recorded at the screening visit.

Pregnancy Test

All panelists (with the exception of post-menopausal women) were given aurine pregnancy test at the screening visit.

Dermal Evaluations

In order to determine the effects of treatment on the skin, a CRLTechnician performed dermal evaluations on each subject's face toexamine for the presence of erythema, edema and dryness. The extentand/or severity of evidence of dermal irritation was recorded utilizingthe following scoring scale:

Scoring Scale for Erythema, Edema and Dryness

0=None 1=Mild 2=Moderate 3=Severe

Efficacy Evaluations

Cutometer

The suction is generated by a variable vacuum pump, and the depth ofpenetration of the skin into the probe is measured optically with anaccuracy of 0.01 mm. The probe is attached to a computer, which controlsthe vacuum application and plots skin deformation as a function of time.From the resulting curve, a number of variables can be extrapolatedincluding immediate, delayed and final distention and immediateretraction (Elsner 1990).

These variables estimate the elastic, viscoelastic and purely viscousbehavior of the skin (Barel et al. 1995).

The following parameters were measured:

The final distention, Uf, measured at 10 seconds. The immediatedistention, Ue, measured at 0.1 seconds. The delayed distention, Uv.Immediate retraction, Ur

The deformation parameters are extrinsic parameters dependent on skinthickness.

In order to circumvent the measurement of skin thickness, the followingratios were used to evaluate the elastic nature of the skin (Elsner1990, Cua et al. 1990, Krushe and Wonet 1995 and Dobrev 1995):

Ur/Ue is the biological elasticity of the skin. It measures the abilityof the skin to regain its initial configuration after deformation. Avalue of 1 indicates 100% elasticity.

Uv/Ue is the ratio between delayed and immediate deformation, i.e., itis the viscoelastic to elastic ratio. An increase in the value of thisratio indicates that there has been an increase in the viscoelasticportion of the deformation and/or relative decrease of the elastic part.

Ur/Uf is a measure of the net elasticity of the skin.

This study consisted of a pre-screening visit, during which timesubjects were examined by a Clinical Research Technician to determineeligibility. Eligible subjects exhibited mild to moderate fine lines andwrinkles on the crow's feet area of the face and experienced chronicmuscle and/or joint pain. At this time, subjects were informed not toeat or drink anything after 9:00 p.m. on the evening prior to thescheduled screening visit, in preparation for the blood and urineanalysis.

Prior to study initiation, subjects arrived at the CRL laboratory forthe scheduled screening visit. Blood was drawn from each perspectivesubject and the forearm utilized for blood collection was recorded on acase report form. A urine specimen was obtained from each panelist forurine analysis, an assessment of sitting vital signs (i.e., pulse, bloodpressure and respirations) was performed and recorded, and all panelists(with the exception of pre-menopausal subjects) were given a urinepregnancy test. Subjects discontinued the use of all oral supplementsfor the remainder of the conditioning period and for the duration of thestudy, with the exception of the oral supplements provided to eachsubject during the study.

To standardize the skin condition of the study population at baselineand minimize variability attributable to use of different skin careregimens, a one week conditioning period began the study. Subjects wererequired to undergo a one week conditioning period, during which time aglycerin soap was used for all facial washing prior to study initiation.At the screening visit, each subject was provided with detailed studyinstructions, a bar of glycerin soap to use for all facial washing,Cetaphil® Daily Facial Moisturizer SPF 15 to use once per day, and aDaily Diary to record soap usage and verify compliance. Subjects begancleansing the face with the soap twice daily (morning and night),beginning seven days prior to the baseline visit. At this screeningvisit, every subject completed a screening questionnaire.

Qualified subjects had normal blood and urine test results and eachpanelist's vital signs were within the normal range, as a result of thepreviously mentioned screening visit safety evaluations. Additionally,eligible subjects who agreed to follow-up visits and study requirementsand met all of the Inclusion/Exclusion Criteria, were to be impaneledfor the study.

Upon returning to the CRL testing facility for the baseline visit,subjects acclimated to ambient laboratory conditions for a period of 30minutes prior to evaluations. Following the acclimation period, a dermalevaluation of the face of each subject was performed by a trained CRLTechnician, for the presence of erythema, edema and dryness. For eachsubject, either the right or the left side of the face was selected formeasurement, as determined by a computer-generated randomization code(FIG. 4). Measurement designation remained consistent throughout thestudy. Cutometer readings were captured from the cheekbone region belowthe eye on the same side of the face. All measurements wereappropriately recorded. Each subject completed a baseline consumerperception questionnaire in order to evaluate the type and frequency ofmuscle and joint pain.

A specified test material was randomly assigned to each subject, suchthat Group 1 panelists received the active tablets, and Group 2 receivedthe placebo tablets. Subject group designations were determined inaccordance with a computer-generated randomization code. Treatmentdesignation/group assignment remained consistent throughout the study(FIGS. 5-6).

Each subject was provided with a designated test material, detailedstudy instructions and a new Daily Diary in which to record daily timesof supplement use, daily application of the Cetaphil® Daily FacialMoisturizer SPF 15, and any suggestive comments pertaining to supplementusage.

The printed study instructions detailed study procedures for each visitand the appointment dates and times of the return visit(s) to ClinicalResearch Laboratories, Inc. Subjects were instructed to continue the useof the provided glycerin soap, twice daily, as well as the use of theCetaphil® Daily Facial Moisturizer SPF 15, once per day.

Each subject was contacted, via telephone, by a CRL Technician for aWeek 4 compliance check. After 8 weeks of ingesting the designated oralsupplement, subjects reported to the CRL testing facility and eachpanelist acclimated to ambient temperature and humidity for a period of30 minutes. Following acclimation, a dermal evaluation of the face ofevery subject was performed by a trained CRL Technician. Cutometerreadings were taken. These procedures and evaluations were performed andrecorded, as previously explained for the baseline study interval.

Subjects reported to Clinical Research Laboratories, Inc. following 12weeks of supplement use and each subject acclimated to ambienttemperature and humidity for a period of 30 minutes. Blood was drawnfrom each subject and the forearm used for blood collection was recordedon a case report form. A final urine specimen was obtained from eachpanelist. A dermal evaluation of the face was performed by a trained CRLTechnician, for the presence of erythema, edema and dryness. Cutometerreadings were captured from the cheekbone region below the eye on thesame side of the face. All measurements were appropriately recorded.

At Week 12, all subjects completed a final questionnaire and unusedportions of test materials and Daily Diaries were returned.

Statistical Methods

The primary efficacy parameters yielding significant results for thisstudy are Cutometer measurements and consumer perception questionnaires.

One-way ANOVA tests (repeat-measurement) were applied to each group todetermine if there were differences between baseline and Week 8, andbaseline and Week 12 for all the parameters. If overall F-test wassignificant, Tukey's multiple comparison test was further applied todetermine where the significant differences exist. The ANOVA test usesthe F-test to determine whether there exists a significant differenceamong means of treatments or time intervals. When the F test rejects thenull hypothesis, it is usually necessary to undertake a thoroughanalysis of the nature of the factor-level effects. Tukey test is amultiple comparison procedure. This is the method which examines orcompares more than one pair of means or proportions at the same time butcontrols the overall significance level to be the same as that which isspecified for a single pair comparison.

The comparisons between Group 1 (subjects assigned to the invention testmaterial supplement) and Group 2 (subjects assigned to the placebo testmaterial supplement) were based on percent changes from baseline at eachtime interval (Week 8 and Week 12). Two-sample t-tests were applied todetermine if significant differences exist between Group 1 and Group 2.(FIGS. 8A-8D)

Two-tailed p-values of 0.05 or less was the criteria for significance,corresponding to a confidence level of 95%. All statistical computationswere performed using Microsoft Excel or SAS® Statistical AnalysisSoftware.

Results

Twenty subjects completed the study. One subject (#7) discontinued studyparticipation for reasons unrelated to the test material.

Adverse Event

On Nov. 20, 2006, subject #6 (assigned to the placebo product) reportedthat on Nov. 17, 2006, following approximately 9 weeks of ingesting theplacebo tablets, she noticed a red rash and hive-like bumps in the cheekarea on both sides of her face and on her upper chest. The subject alsoreported that she experienced itching of these areas. On Nov. 20, 2006,upon examination by a CRL Certified Dermatologist, the subject wasdiagnosed with poison ivy. Subject #6 continued study participation. TheInvestigator determined that this adverse event of moderate severity wasabsolutely not related to the use of the test material. Follow-uptelephone calls were made by a CRL Technician, and the information fromeach conversation was documented as follows: On Nov. 22, 2006, thesubject reported that the hives had disappeared and the facial rednesswas diminishing.

On Nov. 27, 2006, subject #6 indicated that the rash was drying andnearly all of the redness was gone. On Dec. 8, 2006, the subjectreported that the poison was gone and the adverse facial skin conditionhad completely resolved.

Instrumental Measurements, Dermal Evaluations And QuestionnaireResponses

For both groups of subjects, FIGS. 7A-D summarize the Cutometermeasurements for each panelist at each designated study interval. TableVI lists the statistical variables determined for comparisons of thedifferences between Week 8 and Week 12 measurements to baseline. Foreach subject, Table VII lists the baseline, Week 8 and Week 12 dermalevaluation scores for the presence of erythema, edema and dryness. TableVIII lists the questionnaire data for the screening questionnaire[combining both groups of subjects] based on percentages. Tables IX andX list the questionnaire data for the baseline and Week 12 (final)consumer perception questionnaires for Group 1 (subjects assigned to theactive tablets) and for Group 2 (subjects assigned to the placebotablets) based on percentages. Open-ended questions are also included inthese questionnaire tables.

Conclusion

For each subject, study procedures included blood and urine analyses,sitting vital signs, a pregnancy test (with the exception ofpre-menopausal panelists), Cutometer measurements and consumerperception questionnaires.

Blood Analysis

For each subject, a blood analysis was performed at the baseline andWeek 12 study intervals, as indicated above. No clinically significantfindings were determined at Week 12 relative to that which was revealedat baseline.

Urine Analysis

A urine specimen was obtained from each subject at baseline and at theWeek 12 study interval, as specified above. No clinically significantfindings were determined at Week 12 relative to that which was revealedat baseline.

Efficacy Evaluations

As assessed by the Cutometer parameter Ur/Ue, statistically significantdifferences existed between baseline and Week 12 for Group 1 subjects(assigned to the raw ingredient test material supplement, 10853LLA). TheCutometer parameter Ur/Ue describes skin elasticity as it relates tostretching and contracting. Seventy percent of Group 1 subjects showedan improvement in Cutometer parameter Ur/Ue. Relative to baseline, thissignificant increase in parameter Ur/Ue is equivalent to a 29%improvement at Week 12, suggesting that skin elasticity anddistensibility improved for Group 1 subjects who ingested 10853LLA, theinvention supplement. FIGS. 7A-8D summarize the statistical analysis ofstudy results. FIG. 7D show a statistically significant improvement ofUr/Ue from baseline to week 12 (p=0.0109)

Questionnaire responses which were analyzed via Z-score analysisconsisted of question numbers 10 through 15 in the Week 12 (Final)questionnaire. Favorable consumer perception questionnaire responses ofthe study population at Week 12 (final visit), which were applicable toZ-score analysis, as listed below, were measured with statisticalsignificance (Z-scores greater than or equal to the absolute value of1.96) in questionnaires completed by each subject.

A significant proportion of the Group I study population assigned to10853LLA, the active tablets, perceived improvements following 12 weeksof supplement use and subjects responded favorably with regard to thefollowing treatment effects:

Approximately 85% of subjects agreed that after using the product for 12weeks, they experienced muscle and/or joint pain less frequently.

Approximately 85% of subjects agreed that after using the product for 12weeks, the overall pain has been less intense.

Approximately 90% of subjects agreed that after using the product for 12weeks, the pain did not last as long.

Approximately 85% of subjects agreed that the product had a positiveeffect on their joint and/or muscle pain. The detailed statisticalresults are shown in FIGS. 8A-B.

For the Group 2 study population assigned to 10853LLB, the placebotablets, no favorable responses were reported following 12 weeks ofsupplement use. The results are shown in FIGS. 9A-B.

STUDY REFERENCES

-   Basel, Courage and Clarys, Handbook of Non-Invasive Methods and the    Skin, Serup and Jemec, eds. 1995.-   Blichman and Serup, Acta Derm Venereol, 1988.-   Cua, Maibach and Wilhelm, Dermatologica Research, 1990.-   Dobrev, Sixth Congress of Dermatology and Venerology, Pleve,    Bulgaria, May 11-13, 1995.-   Elsner, Eighth International Symposium “Bioengineering and the    skin,” Stresa/ltalia, June 1990.-   Frankowski, Chen and Huth, Photonics West Annual Symposium,    Electronics Imaging 2000, San Jose, January 23-28, 2000.-   Fiorentini, Becheroni, Iorio. Int. J. Cosmet. Sci. 26-29. 1988.-   Friedman, Skover, Payonk and Geronemus, Seminars in Cutaneous    Medicine and Surger, 200, 21 (4):266-273.-   Krushe and Worre, Archives of Dermatologica Research. 287-293, 1995.-   Morganti, Randazzo and Cardillo. Appl. Cosmet. 10-12, 1986.-   Randeau, Kurdian, Sirvent, Closs and Girard, Cosmetics and    Toiletries Manufacture Worldwide 2002.-   Simon, Arzliche Kosmetologie, 256-259, 1984.

All references mentioned above are expressly are expressly incorporatedby reference into the instant application.

Method for Monocyte Activation Assay

Monocyte activation assay was used to evaluate immunostimulatoryactivity. The THP-1 human monocyte cell line was transfected with aluciferase reporter gene construct containing two copies of the NF-kappaB motif for HIV/IgK as described previously. This monocyte activationassay is an example of an in vitro test system that can be used forbioactivity based standardization of Aloe vera extracts and productmaterial.

Method for Toll-Like Receptor 2 (TLR2) Expression Vector Experiment

HEK 293 cells were cultured in DMEM/F12 medium supplemented with fetalbovine serum (10%) and 1% penicillin-streptomycin at 37° C., under 5%CO₂. Actively growing cells were transiently transfected with theappropriate plasmid(s) using electroporation (at 150 V and one 70-mspulse). Following electroporation, cells were plated at a density of5×10⁴ cells in 200 μl/well of culture medium. After 48 hours, agents tobe tested were added to transfected cells. Six hours after addition ofsamples, cells were harvested and luciferase activity measured. TheNF-kappa B plasmid construct (pBIIXLUC) contained two copies of NF-kappaB motif from HIV/IgK. Plasmids co-expressing human CD14 and human TLR2(pDUO-hCD14/TLR2) were purchased from InvivoGen. Zymosan was purchasedfrom Sigma, Staphylococcus aureus peptidoglycan from Fluka and ultrapure Salmonella minnesota LPS from List Biological Laboratories, Inc.

AloeEx Activates NF-Kappa B In Vitro Through a TLR2-Dependent Process.

FIGS. 10A-10B depict that AloeEx (batch 2-5-05) acts through TLR2. HEK293 cells (cell line with minimal TLR expression) were transientlytransfected with a luciferase reporter gene construct containing twobinding sites for NF-kappa B, alone (FIG. 10A), or in combination withthe pDUO-hCD14/TLR2 expression plasmid (FIG. 10B). The pDUO-hCD14/TLR2expression plasmid expresses proteins supporting TLR2-(CD14 and TLR2)dependent NF-kappa B activation. Forty-eight hours after transfection,cells were treated with either AloeEx TLR2 agonists peptidoglycan (PG)or zymosan (Z), or TLR4 agonist ultrapure S. minnesotalipo-polysaccharide (LPS) at the indicated concentrations (μg/ml).Luciferase activity was determined 6 hours after addition of agents.

Neither the TLR2 specific (peptidoglycan or zymosan) or TLR4 specific(ultrapure LPS) ligands nor AloeEx activated NF-kappa B-dependentluciferase expression in HEK 293 cells transfected with the reporterplasmid alone (FIG. 10A). AloeEx activated NF-kappa B in cellssupporting TLR2-dependent (expressing CD12 and TLR2) activation (FIG.10B). Peptidoglycan and zymosan also activated NF-kappa B in cellsexpressing TLR2 and CD14 as compared with ultrapure LPS which did not.

Presence of Alps and Alp in AloeEx

The inventors have demonstrated that AloeEx contains bacterial LPS andbacterial lipo-proteins that are most likely derived from microorganismsthat are associated with Aloe vera. The presence of these bacterialcomponents in AloeEx are believed to contribute to the therapeuticproperties of products derived from AloeEx.

AloeEx was extracted with 90% aqueous phenol: water (1:1) at 70° C. Uponcooling the extract and subsequent centrifugation two layers were formed(water and phenol) and a pellet of insoluble material. The water layercontains ALPS. Due to the non-polar lipid moiety on lipo-proteins, thephenol layer contain the ALPs. Using this procedure, AloeEx (batch 056)was separated into a water layer fraction, a phenol layer fraction andinsoluble material. The water layer fraction was freeze-dried (8.9%yield). Material in the phenol layer was precipitated by addition of 2volumes of ether:acetone (1:5) and 4 volumes of ethyl acetate. Thephenol layer precipitate was washed extensively with ethyl acetate andisopropanol and then dried at 60° C. (45% yield). The original AloeExmaterial, water layer fraction, phenol layer fraction and insolublematerial were all tested in the monocyte assay (FIG. 11). Twenty-fourshours following transfection with the NF-kappa B luciferase reporterplasmid, cells were treated with the indicated sample for 4 hours(concentrations in μg/ml). Luciferase activity was determined and isreported as a percent of maximal light output from LPS-treated cells.Both the water layer and phenol layers exhibited substantial monocyteactivation believed to be due to bacterial ALPS and ALP, respectively.The insoluble material was also active as indicated by its ability toenhance NF-kappa B activation by 35% when tested at 5 μg/ml.

Characterization of Alps in AloeEx Phenol Layer Fraction

The phenol layer fraction material was extracted three times with 4%sodium dodecyl sulfate (SDS) (26 mg/ml) at 100° C. for 30 minutes. SDSinsoluble material was removed by centrifugation and when tested in themonocyte assay contained only about 1-2% of the total activity presentin the original phenol layer fraction.

The SDS extract of the phenol layer fraction was incubated with 0.1mg/ml (3.9 units/ml) proteinase K from Tritirachium album (Sigma) in 50mM TRIS (pH 7.5), 5 mM ß-mercaptoethanol, and 5 mM CaCl₂ for 2 hours at50° C. The digest was then heated at 98° C. for 10 minutes. A controlsample, without proteinase K, was run under identical conditions. ForSDS polyacrylamide gel analysis, extract (proteinase K treated anduntreated) was mixed with 1 volume of Tris-Tricine sample buffer(Bio-Rad) and loaded in nonadjacent lanes of a 16.5% Tris-Tricineprecast gel (Ready Gel, Bio-Rad). The amount of sample loadedrepresented the SDS extractable material present in 130 μg of AloeExphenol layer fraction. Wide molecular weight range protein marker(Sigma) was run in the gel. Individual gel lanes were cut into 12 equalsections (0.5 cm/section) and extracted with 150 μl of 1.5%octylglucoside, 2.5 mM TRIS (pH 7.5), 5 mM CaCl₂ at 95° C. for 5minutes. Supernatants of samples were collected and evaluated foractivity in the monocyte activation assay.

FIG. 12 indicates that protein does not appear to be directlyresponsible for the activation of the monocytes by AloeEx phenol layerfraction, but rather that protein is part of the molecule that isresponsible for this activity. This is indicated by the reduction in theapparent size of the active compounds following proteinase K treatmentas determined by fractionation on an SDS-polyacrylamide gel. Followingproteinase K treatment there was a substantial reduction in activity forthe region between 66 and 6.5 kDa, while there was an increase inactivity for the region between 6.5 kDa and the gel front. This resultis similar to those obtained with bacterial lipoproteins in thatproteinase K digestion does not reduce the activity of the lipoproteins(i.e. the protein component of the lipo-protein is not necessary formonocyte activation) but proteinase K treatment does reduce the size ofthe lipo-proteins when fractionated on an SDS-polyacrylamide gel. Theblack bars labeled “C” and “PK” in FIG. 12 represent the activity of theuntreated and proteinase K treated AloeEx phenol layer fraction,respectively, before fractionation on the gel.

To determine sensitivity of the 4% SDS extract of the AloeEx phenollayer fraction to lipo-protein lipase, the SDS was removed using SDS-outreagent (Pierce) in the presence of 0.88% octylglucoside. The sample wasthen adjusted to a final concentration of 10 μM AEBSF protease inhibitorcocktail solution (Sigma) and 0.2% BSA (Sigma, No. A-9418). The samplewas incubated at 37° C. for 16 hours with 39,600 units/ml (1 mg/ml) oflipo-protein lipase from Pseudomonas species (Sigma No. L9656). Controlsamples (without lipo-protein lipase) were run under identicalconditions. Activity of lipo-protein lipase treated and untreatedsamples were evaluated using the monocyte activation assay.

The results as depicted in FIG. 13 show that the activity present in the4% SDS extract of AloeEx phenol layer fraction is completely abrogatedby treatment with lipo-protein lipase. This result together with theresults presented in. FIG. 12 confirms that the activity in the AloeExphenol layer fraction is due to ALPs. This procedure demonstrated thatAloeEx contains ALPs that are believed to be derived from microorganismsthat are associated with Aloe vera. The presence of this bacterialcomponent, combined with other components in AloeEx, are believed tosignificantly contribute to the therapeutic properties of this product.

Characterization of Alps in AloeEx

The following summarizes the amount of ALPS present in AloeEx (batch056) and the two fractions derived from this material (water layerfraction and phenol layer fraction).

AloeEx (batch 056) 625-6250 EU/mg

AloeEx Water Layer Fraction 62,500-625,000 EU/mg

AloeEx Phenol Layer Fraction <4.8 EU/mg

The amount of ALPS was estimated using Pyrosate LAL assay kit(Associates of Cape Cod Inc) according to manufacture's instruction. Asexpected, the water layer fraction contained essentially all of the ALPSpresent in AloeEx, as compared to the phenol layer fraction. This datademonstrated that AloeEx contains ALPS that is believed to be derivedfrom microorganisms that are associated with Aloe vera. The presence ofthis bacterial component combined with other components in AloeEx arebelieved to contribute to the therapeutic properties of this product.

The Rind and Mucilage Contain the Majority of the Monocyte ActivatingComponents in Aloe Vera

The Aloe vera leaf can be divided into three basic structural parts: thegel, the outer rind and the mucilage (the layer between the gel and theouter rind). An experiment was conducted to determine which part of theAloe leaf contained the highest level of monocyte activating components.From one Aloe vera leaf, the gel was carefully cut-out by passing aknife parallel to the inner rind surface. After removal of the gel, theleaf was scraped over the edge of a vial to collect the mucilage partand to separate it from the outer rind. All three components and asection of the whole leaf Aloe were freeze-dried and ground to a finepowder. Each sample was then extensively extracted with methanol toremove non-polar substances, such as anthraquinones, that have aninhibitory effect in the monocyte activation assay. Samples withnon-polar substances removed were prepared as a fine suspension inwater. Twenty-fours hours following transfection with the NF-kappa Bluciferase reporter plasmid, THP-1 cells were treated with the preparedsamples for 4 hours. Luciferase activity was determined and is reportedas a percent of maximal light output from ELPS-treated cells. FIG. 14depicts the response of the different Aloe vera leaf parts and that ofthe whole leaf material. This data suggests that the mucilage layer andouter rind contain the majority of the monocyte activating components inthe Aloe leaf. In this experiment, the percent that each leaf partcomprises of the total dry weight of the Aloe leaf was: gel (8.7%),mucilage layer (6.2%) and rind (85.1%).

Since the outer rind and mucilage layer contain the majority of themonocyte activating components, and these parts also comprise themajority of the dry solids in the Aloe leaf, the inventors conclude thatthe outer rind and mucilage layer are of commercial interest. Typically,the outer rind is discarded as a waste product after the gel has beenremoved or after the whole Aloe leaf has been processed to create Aloejuice. Another waste product is Aloe pups (small Aloe plants that growoff of the larger plants). Since the Aloe pups contain mostly rind withvery little gel and are considered of little commercial value. The rindand Aloe pups waste products can be processed to produce for example,dietary supplements that enhance immune function. The dried rind couldbe ground to a powder and processed to remove anthraquinones. Thismaterial could be further processed by extraction to produce crudeextracts and purified fractions. The objective of the crude extracts andpurified fractions would be to concentrate the amount of AloeEx and/ormicroorganisms and microorganism fragments/components associated withAloe vera.

Monocyte Activating Components in AloeEx are Extractable and Soluble inDetergent

For development of various products (e.g. for cosmetics) it may bedesirable to solublize the immune enhancing components in AloeExmaterial. The immune enhancing components would include Aloeridepolysaccharide, ALPS and ALPSs derived from microorganisms that areassociated with Aloe vera. As previously described, a significantportion of the immune enhancing components of AloeEx material can befractionated into a water layer fraction and phenol layer fraction. Theimmune enhancing activity present in the phenol layer fraction (ALPS)can be completely extracted/solubilized with 4% SDS at 100° C. Theimmune enhancing activity present in the water layer (ALPS and Aloeridepolysaccharides) is water soluble and also SDS soluble.

Extraction of AloeEx with SDS represents a potential method to solublizea significant portion of the immune enhancing components present in thismaterial. The data suggest that other known detergents (including thosethat are more compatible with product formulation) could also bepotentially useful in extraction/solublization of the immune enhancingcomponents in AloeEx material. This method of detergent extraction couldalso be used to solubilize the monocyte activating components (i.e.Aloeride polysaccharide and microorganism components associated withAloe vera) from raw materials such as whole leaf, rind, Aloe pups, etc.

Method for Producing an Extract by Isolation of Microorganisms andMicroorganism Components/Fragments Associated with Aloe Vera

As demonstrated, bacterial products from microorganisms associated withAloe vera are responsible for a significant portion of the immuneenhancing properties of AloeEx. It is shown that AloeEx accounts for amajority of the in vitro monocyte/macrophage activation potential ofAloe vera. Therefore, it is believed that bacterial products frommicroorganisms are responsible for a signifcant portion of the immuneenhancing properties of Aloe vera.

Extracts that concentrate the amount of microorganisms and/ormicroorganism components/fragments associated with Aloe vera can beprepared from crude Aloe material and/or Aloe vera raw material,including waste material (e.g. leftover rinds, Aloe pups, etc.). Crudeextracts can be processed to create purified fractions by extracting thebacterial lipo-proteins and/or other bacterial components using asolvent. These fractions would contain enhanced levels of bacterialcomponents associated with Aloe vera. Solvents which may be used toprepare a purified fraction include detergents such as SDS, water, watermiscible solvents such as alcohol, and combinations of these solvents orother solvents well known in the art.

The following procedure could be used to prepare an extract thatconcentrates the microorganisms and/or microorganismcomponents/fragments associated with Aloe vera. Aloe vera raw material(gel, whole leaf, rind, pups, etc.) can be liquified using methods knownin the art. The liquid can be subjected to coarse filtration to removefibrous material. A food-grade flocculant (clarifier or similarsubstance) can be added to the liquified Aloe to create aggregates ofparticles (bacterial, other microorganisms, microorganismfragments/components). These aggregrated particles could then beseparated from the remaining liquid using methods known in the art. Suchmethods would include centrifugation, filtration, settling followed bydecanting, filtering aggregates out of the liquid using a net, etc. Thefinal collected material can be also processed to remove anthraquinones,if present, using a technique such as washing/extracting with alcohol.If desired, the resulting material can be dried.

Method for Producing a Standardized Aloe Vera Product

Biological and chemical standardization represent two methods that canbe used to standardize Aloe vera products that contain the immuneenhancing agents described in the present invention. Both methods can beused for standardizing either extract material or the raw material. Thepurpose of standardization is to ensure that each batch of productmaterial contains the same level of active component(s).

In one method a bioactivity standardized Aloe vera product containing aneffective amount of immunostimulatory activity is prepared. In thismethod, Aloe vera product material is tested in vitro for activation ofimmune cells and the bioactivity is then compared to a standardpreparation immunostimulatory value to determine a standardized activityvalue of the product. An example of an in vitro test system is themonocyte activation assay previously described. Bioactivity basedstandardization of product material is important when the chemicalcontent of the active components do not correlate with biologicalactivity due to unknown structure-activity relationships and/or complexinteractions between multiple actives. Under such circumstances, theamount of active substances is not sufficient to reflect the potency ofthe product material and standardization through the use of a biologicalassay is desirable. Bioactivity standardization has been used by thepharmaceutical industry for biologics such as insulin and cytokines.

In another method a chemically standardized Aloe vera product containingan effective amount of immunostimulatory lipo-proteins originating frommicroorganisms associated with Aloe vera is prepared. A chemical markerwhich may be used for standardization is 2,3-dihydroxypropyl cysteine.This modified cysteine amino acid is thought to be unique tolipo-proteins that are immunostimulatory from prokaryotic organisms. Inthis method, Aloe vera product material is tested for the amount of2,3-dihydroxypropyl cysteine and then compared to the amount of2,3-dihydroxypropyl cysteine in a standard preparation to determine astandardized value of the product.

In certain embodiments a food grade flocculent, clarifier or coagulantmay be used to form microorganism aggregates. Examples include, but arenot limited to, for example, alum, aluminium chlorohydrate, aluminumsulfate, calcium oxide, iron(III) chloride, iron(II) sulfate,polyacrylamide, sodium aluminate, sodium silicate and combinationsthereof. Natural products which can also be used as flocculants includeChitosan, Moringa oleifera seeds, Papain, species of Strychnos (seeds)Isinglass and combinations thereof. Other such materials are well knownto those skilled in the art.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of the principles and applications of thepresent invention. It is therefore to be understood that numerousmodifications may be made to the illustrative embodiments and that otherarrangements may be devised without departing from the spirit and scopeof the present invention as defined by the appended claims.

1. (canceled)
 2. An immunostimulatory composition comprising: aneffective amount of an immunostimulatory Aloe vera-derived mixture andan Aloe vera-derived material, wherein the immunostimulatory Aloevera-derived mixture comprises at least one Aloe vera-derivedlipo-polysaccharide, at least one Aloe vera-derived polysaccharide andat least one Aloe vera-derived lipo-protein, wherein theimmunostimulatory Aloe vera-derived mixture exhibits animmunostimulatory activity of at least 50% of a maximal NF-kappa Betadirected luciferase expression by Escherichia coli bacteriallipo-polysaccharide (ELPS) at a concentration of about 10 μg/mL, and theimmunostimulatory activity is measured by NF-kappa Beta directedluciferase expression in THP-1 monocytes at a concentration of about 250μg/mL or less, wherein the Aloe vera-derived material is selected fromthe group consisting of Aloe vera gel concentrated at 1×, Aloe vera gelconcentrated at 10×, Aloe vera gel concentrated at 200×, Aloe vera wholeleaf spray dried powder concentrated at 100×, Aloe vera whole leafdecolorized liquid, acemannan, and Aloe vera mucilaginouspolysaccharide, wherein the amount of the Aloe vera-derived materialranges from 10 wt % to about 90 wt % relative to the total amount of theimmunostimulatory Aloe vera-derived mixture and the Aloe vera-derivedmaterial, and wherein the composition is selected from the groupconsisting of a dispersion, a solution, and a suspension.
 3. Theimmunostimulatory composition of claim 2, wherein the Aloe vera-derivedpolysaccharide comprises glucose in molar amount ranging from about 25%to about 70%, rhamnose in a molar amount ranging from about 0.5% toabout 35%, galactose in a molar amount ranging from about 5% to about30%, mannose in a molar amount ranging from about 3% to about 25%, andarabinose in a molar amount ranging from about 0% and about 15%, basedon the total molar amount of the Aloe vera-derived polysaccharide, andwherein the Aloe vera-derived polysaccharide has a molecular weightgreater than about 2,000,000 Daltons.
 4. The immunostimulatorycomposition of claim 3, wherein the immunostimulatory Aloe vera-derivedmixture comprises between 0.01% and 50% by weight of the Aloevera-derived polysaccharide and between 0.0001% and 10% by weight of thecombination of the Aloe vera-derived lipo-protein and Aloe vera-derivedlipo-polysaccharide.
 5. The immunostimulatory composition of claim 4,wherein the immunostimulatory Aloe vera-derived mixture comprisesbetween 1% and 20% by weight of the Aloe vera-derived polysaccharide. 6.The immunostimulatory composition of claim 4, wherein theimmunostimulatory Aloe vera-derived mixture comprises between 1% and 15%by weight of the Aloe vera-derived polysaccharide.
 7. Theimmunostimulatory composition of claim 4, wherein the immunostimulatoryAloe vera-derived mixture comprises between 10-15% by weight of the Aloevera-derived polysaccharide.
 8. The immunostimulatory composition ofclaim 2, wherein the immunostimulatory Aloe vera-derived mixture isobtained from Aloe vera, wherein the Aloe vera includes at least one ofAloe vera rind, Aloe vera mucilage, Aloe vera gel, or whole leaf Aloevera.
 9. The composition of claim 2, further comprising: apharmaceutical carrier or excipient.
 10. The immunostimulatorycomposition of claim 2, wherein the Aloe vera-derived material isselected from the group consisting of an immunostimulatory Aloevera-derived material and a non-immunostimulatory Aloe vera-derivedmaterial.
 11. The immunostimulatory composition of claim 2, wherein theAloe vera-derived material is Aloe vera gel concentrated at 200× andwherein the Aloe vera gel concentrated at 200× is present in thecomposition in an amount of about 80% by weight relative to the totalamount of the Aloe vera-derived material and the immunostimulatory Aloevera-derived mixture.
 12. A method of treating muscle or joint pain in asubject in need thereof comprising administering to said subject aneffective amount of the immunostimulatory composition of claim
 2. 13.The method of claim 12, wherein the subject is a human being.
 14. Themethod of claim 12, wherein the effective amount of theimmunostimulatory composition is between 0.2 milligrams and 1 gram perday.
 15. A method of improving the appearance, texture, firmness and/orelasticity of skin and/or for providing accelerated growth of new skincells in a subject in need thereof comprising administering to saidsubject an effective amount of the immunostimulatory composition ofclaim
 2. 16. The method of claim 15, wherein the subject is a humanbeing.
 17. The method of claim 15, wherein the effective amount of theimmunostimulatory composition is between 0.0005 milligrams and 1 gramper day.
 18. The immunostimulatory composition of claim 2, wherein thetotal amount of the immunostimulatory Aloe vera-derived mixture and theAloe vera-derived material is present in the immmunostimulatorycomposition in an amount between 0.05% and 5% by weight of the totalweight of the immunostimulatory composition.
 19. The immunostimulatorycomposition of claim 18, wherein the total amount of theimmunostimulatory Aloe vera-derived mixture and the Aloe vera-derivedmaterial is 0.5% by weight of the total weight of the composition.
 20. Amethod of treating muscle or joint pain in a subject in need thereofcomprising administering to said subject an effective amount of theimmunostimulatory composition of claim
 11. 21. A method of improving theappearance, texture, firmness and/or elasticity of skin and/or forproviding accelerated growth of new skin cells in a subject in needthereof comprising administering to said subject an effective amount ofthe immunostimulatory composition of claim
 11. 22. The composition ofclaim 11, further comprising a pharmaceutical carrier or excipient. 23.A method of treating muscle or joint pain in a subject in need thereofcomprising administering to said subject an effective amount of theimmunostimulatory composition of claim
 22. 24. A method of improving theappearance, texture, firmness and/or elasticity of skin and/or forproviding accelerated growth of new skin cells in a subject in needthereof comprising administering to said subject an effective amount ofthe immunostimulatory composition of claim
 22. 25. An immunostimulatorycomposition, comprising: an effective amount of an immunostimulatoryAloe vera-derived mixture, the immunostimulatory Aloe vera-derivedmixture comprising at least one Aloe vera-derived lipo-polysaccharide,at least one Aloe vera-derived polysaccharide, and at least one Aloevera-derived lipo-protein, wherein the immunostimulatory Aloevera-derived mixture exhibits an immunostimulatory activity of at least50% of a maximal NF-kappa Beta directed luciferase expression byEscherichia coli bacterial lipo-polysaccharide (ELPS) at a concentrationof about 10 μg/mL, and the immunostimulatory activity is measured byNF-kappa Beta directed luciferase expression in THP-1 monocytes at aconcentration of about 250 μg/mL or less, and wherein the composition isselected from the group consisting of an dispersion, a solution, and asuspension.
 26. The immunostimulatory composition of claim 25, whereinthe immunostimulatory Aloe vera-derived mixture is insoluble in water.27. The immunostimulatory composition of claim 25, further comprising: acomplexing agent selected from the group consisting ofethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as thedisodium salt, citric acid, nitrilotriacetic acid, and the saltsthereof.
 28. The immunostimulatory composition of claim 25, furthercomprising: an alcohol selected from the group consisting of ethanol,isopropanol, propylene glycol, and polyethylene glycol.
 29. Theimmunostimulatory composition of claim 2, wherein the immunostimulatoryAloe vera-derived mixture exhibits an immunostimulatory activity of atleast 50% of a maximal NF-kappa Beta directed luciferase expression byEscherichia coli bacterial lipo-polysaccharide (ELPS) at a concentrationof about 10 μg/mL, and the immunostimulatory activity is measured byNF-kappa Beta directed luciferase expression in THP-1 monocytes at aconcentration of about 5 μg/mL to about 250 μg/mL.
 30. Theimmunostimulatory composition of claim 2, wherein the immunostimulatoryAloe vera-derived mixture is insoluble in water.
 31. Theimmunostimulatory composition of claim 2, further comprising: acomplexing agent selected from the group consisting ofethylenediaminetetraacetic acid (EDTA) or a salt thereof, such as thedisodium salt, citric acid, nitrilotriacetic acid, and the saltsthereof.
 32. The immunostimulatory composition of claim 2, furthercomprising: an alcohol selected from the group consisting of ethanol,isopropanol, propylene glycol, and polyethylene glycol.